Compositions for lipid matrix-assisted chemical ligation

ABSTRACT

The present invention relates to methods and compositions for lipid matrix-assisted chemical ligation and synthesis of membrane polypeptides that are incorporated in a lipid matrix. The invention is exemplified in production of a prefolded membrane polypeptide embedded within a lipid matrix via stepwise chemoselective chemical ligation of unprotected peptide segments, where at least one peptide segment is embedded in a lipid matrix. Any chemoselective reaction chemistry amenable for ligation of unprotected peptide segments can be employed. Suitable lipid matrices include liposomes, micelles, cell membrane patches and optically isotropic cubic lipidic phase matrices. Prefolded synthetic and semi-synthetic membrane polypeptides synthesized according to the methods and compositions of the invention also permit site-specific incorporation of one or more detectable moieties, such as a chromophore, which can be conveniently introduced during synthesis. The methods and compositions of the invention have multiple uses. For example, they can be used to assay ligand binding to membrane polypeptides and domains comprising a receptor, and thus are extremely useful for structure/function studies, drug screening/selection/design, and diagnostics and the like, including high-throughput applications. The methods and compositions of the invention are particularly suited for FRET analyses of previously inaccessible membrane polypeptides.

Cross-Reference To Related Applications

This Application claims priority to U.S. Pat. applications. Ser. Nos.09/384,302 (filed Aug. 26, 1999. and issued as U.S. Pat. No. 6,451,543on Sep. 17. 2002), 09/263,971 (filed Mar. 5, 1999; abandoned), and09/144,964 (filed Aug. 31, 1998, abandoned).

TECHNICAL FIELD

The present invention relates to membrane polypeptides, methods ofpreparation, and assays that employ them.

BACKGROUND

Cell membranes are made of lipids capable of forming a barrier betweenaqueous compartments. They consist primarily of a continuous double orbilayer plate of lipid molecules associated with various membraneproteins. The phospholipids, sphingolipids, and glycolipids make up thethree major classes of membrane forming lipid molecules. These lipidsare amphipathic (amphiphilic) molecules in that they have a hydrophilic(polar) head and a hydrophobic (non-polar) tail. In the aqueousenvironment of cells, the polar head groups face toward the water whiletheir hydrophobic tail groups interact with each other to create alamellar bilayer, and to a lessor extent other aggregate structuresdepending on the lipid composition and conditions. For example, membranelipids can form a variety of different shapes including spheres(vesicles), rods (tubes) and lamellae (plates) depending on lipid andwater content, and temperature. These shapes represent basic units thatinteract to form two- and three-dimensional lattice matrix structuresclassified as lamellar phase (e.g., bilayer plate, closed sphere),hexagonal phase (e.g., rod), or cubic phase (e.g., spheres, rods orlamellae connected by aqueous channels) (Lindblom, et al., Biochimica etBiophysica Acta (1989) 988:221-256). A cross section of a typical cellmembrane bilayer (lamellar phase) of phospholipid can be viewed ashaving a hydrophobic core region of about 30 Angstroms (Å) with twointerfacial regions of about 15Å each (White, et al., Curr. Struc. Biol.(1994) 4:79-86).

Proteins can associate with cell membranes in different ways. Integralmembrane proteins contain at least one component that is embedded withinthe lipid bilayer. The non-polar segments of these integral membraneproteins, which embed in the lipid bilayer perpendicular to the surfaceof the membrane, may consist of a hydrophobic region of the polypeptide,a covalently attached fatty acid chain or other types of lipid chains.Peripheral membrane proteins normally associate with the lipid bilayerthrough non-covalent interactions with these integral membrane proteins.Additionally, some peripheral membrane proteins are located entirely inthe aqueous phase, associated with the membrane through a covalentlyattached fatty acid or lipid chain. The co-translational attachment of afatty acid chain such as myristic acid to the amino-terminal glycine ofa protein through an amide linkage results in localization of theprotein to the cytoplasmic face of cellular membranes. Prenyl groups andpalmitic acid groups are attached post-translationally via thioetherlinkages to cysteine residues and also result in localization ofproteins to the membrane. These types of covalent attachments areimportant for function in a wide variety of cell signaling proteins,like the heterotrimetric G proteins (James, et al., Biochemistry (1990)29(11):2623-2634; Morello, et al., Biochem. Cell Biol. (1996)74(4):449-457; Mumby, S. M., Curr. Opin. Cell. Biol. (1997)9(2):148-154; Resh, M. D., Cell Signal (1996) 8(6):403-412; and Boutin,J. A., Cell Signal (1997) 9(1):15-35). Glycosylphosphatidylinositolanchors, found at the C-terminus of soluble proteins, result in theattachment of these proteins to the cell surface membrane (Turner, A.J., Essays Biochem. (1994) 28:113-127).

The two major classes of known integral membrane proteins are those thatinsert α-helices into the lipid bilayer, and those proteins that formpores in the lipid bilayer by β-barrel strands (Montal, et al., Curr.Opin. Stuc. Biol. (1996) 6:499-510; Grigorieff,et al., J. Mol. Biol.(1996) 259:393-42; and Weiss, et al., J. Mol. Biol. (1992) 227:493-509).Single membrane spanning proteins, or single-pass membrane proteins,generally have a hydrophobic region that anchors that sequence in thelipid bilayer via an α-helix configuration. Multiple membrane spanningproteins, or multi-pass membrane proteins, result from the polypeptidechain passing back and forth across the lipid bilayer and typicallyemploy cc-helix and/or β-barrel structured membrane anchors.

Examples of membrane proteins include membrane-associated receptors,transporter proteins, enzymes, and immunogens. For instance, cellmembrane-associated receptors represent a dynamic collection of membraneproteins of particular therapeutic importance. Four basic superfamiliesare recognized: the enzyme-linked receptors, the fibronectin-likereceptors, the seven transmembrane receptors, and the ion channelreceptors. Enzyme-linked receptors represent single-pass membraneproteins, with the basic structure consisting of a single polypeptidetraversing the plasma lamella once via an α-helix anchor domain. Theextracellular domain of enzyme-linked receptors binds hormone/ligand,while the carboxyl-terminal domain contains a catalytic site thatpromotes signal transduction via hormone/ligand binding and receptoraggregation.

The fibronectin-like receptors have the same general structure as theenzyme-linked receptors except that no specific catalytic site isrepresented in the cytoplasmic domain. Class 1 fibronectin-likereceptors contain two modified extracellular domains formed from twoseven stranded β-sheets that join at right angles to a ligand-bindingpocket. The class 2 fibronectin-like receptors have a slightly differentstructure in that they form repeats of five-stranded β-sheets thatextend over the hormone like fingers. The class 1 and 2 receptorscontain a conserved proline-rich cytosolic juxtamembrane region thatconstituatively binds soluble tyrosine kinases, which is activated byligand/hormone-binding and receptor aggregation.

The seven-transmembrane receptors, also called G-protein coupledreceptors, serpentine receptors, or heptahelical receptors, representthe largest and most diverse family of membrane receptors identified todate. These receptors mediate sensory and endocrine related signaltransduction pathways and are multi-pass membrane proteins havingα-helical anchor regions that transverse the membrane seven times. Thetransmembrane spanning regions for some of these proteins form a smallligand/hormone-binding pocket, while larger binding sites are formedthrough extended amino terminal regions. Seven-transmembrane receptorsalso contain one or more intracellular loops that bind and activateG-proteins, which act as second messengers in cells.

The ion channel receptors are represented by the ligand- andvoltage-gated channel membrane protein receptors. Ligand-gated ionchannels are formed by pentamers of homologous subunits. Each subunitcontributes an α-helix toward forming the wall of the channel.Ligand/hormone binding appears to occur between the subunits. Thetypical voltage-gated channel receptors are homotetramers, with eachsubunit having six transmembrane α-helices.

Different techniques have been used to study membrane proteins and/orexploit them for therapeutic purposes, diagnostics, and drug screeningassays and the like. However, unlike non-membrane proteins, the biggestobstacle in working with membrane proteins is the poor solubility oftheir hydrophobic polypeptide chains, the difficulty in folding membraneproteins from unfolded polypeptide chains and the difficulty inoverexpressing and isolating them in environment suitable forquantitative analyses (Huang, et al., J. Biol. Chem. (1981)256:3802-3809; and Liao, et al., J. Biol. Chem. (1983) 258:9949-9955).For example, unfolding and folding whole transmembrane proteins isdifficult since they are insoluble in the lipid bilayer in the unfoldedform, as well as in the aqueous phase in both their folded and unfoldedforms, because of their highly hydrophobic character (Haltia, et al.,Biochimica et Biophysica Acta (1995) 1241:295-322). This feature ofmembrane proteins is particularly problematic when attempting tosynthesize, label or otherwise manipulate them chemically in a cell freeenvironment. Nevertheless, individual transmembrane segments of membraneproteins have been chemically synthesized via solid phase chemistry,followed by subsequent insertion into membranes and spontaneous assemblyof native-like structures with biological activity (Popot, et al.,Biochemistry (1990) 29:4031-4037; and Grove, et al., Methods Enzymol.(1992) 207:510-525). To date, however, solid phase synthesis has beenlimited to synthesis of only a few short transmembrane peptide segments,since membrane proteins are recalcitrant to standard chemical synthesistechniques.

Establishing access to membrane proteins with site-specific chemicalmodifications is crucial both for the analysis of structure-functionrelationships of membrane proteins and for drug discovery. The mostimportant techniques currently employed to achieve this goal are thesynthesis of small membrane-spanning peptide fragments of these proteins(Grove, et al., Methods Enzymology (1992) 207:510-525; and MacKenzie, etal., Science (1997) 276:131-133), chemical modification of existing orengineered cysteine residues (Oh, et al., Science (1996) 273:810-812),and in vitro suppression mutagenesis to incorporate unnatural aminoacids (Cload, et al., Chemistry and Biology (1996) 3:1033-1038; andTurcatti, et al., J. Biol. Chem. (1996) 271:19991-19998). None of thesetechniques provides general access to totally synthetic orsemi-synthetic membrane proteins containing chemically modified aminoacid side-chains, or their production in a quantity sufficient for mostbiophysical techniques. Additionally, such techniques do not permitmodular synthesis and reassembly of membrane-incorporated transmembranepolypeptide segments or domains.

Relevant Literature

Wilken, et al. (Curr. Opin. Biotech. (1998) 9(4):412-426) reviewchemical protein synthesis. Dawson, et al. (Science (1994) 266:776-779)disclose chemical synthesis of water Grove, soluble polypeptides bynative chemical ligation. et al. (Methods in Enzymology (1992)207:510-525) disclose Boc-chemistry solid phase synthesis of small poreforming membrane peptides and their subsequent incorporation andactivity in a lipid membrane. MacKenzie, et al. (Science (1997)276:131-133) disclose recombinant synthesis and radioactive labeling ofthe transmembrane domain of glycophorin A and its incorporation and NMRstructure in a lipid membrane. Oh, et al. (Science (1996) 273:810-812)disclose NMR structure of a diptheria toxin transmembrane domain bychemical modification of existing or engineered cysteine residues with amethanethiosulfate spin label to generate a nitroxide side chain.Turcatti, et al. (J. Bio. Chem. (1996) 271:19991-19998) disclose invitro suppression mutagenesis in Xenopus oocytes to introducefluorescence-labeled amino acids into the seven transmembraneneurokinin-2 receptor and its incorporation and activity in oocytemembranes. Portman, et al. (J. Phy. Chem. (1991) 95:8437-8440) discloseincorporation and activity of α-chymotrypsin and bacteriodopsin in acubic phase lipid matrix. Giorgione, et al. (Biochemistry (1998)37(8):2384-2392) disclose incorporation and activity of protein kinase Cin a cubic lipidic phase matrix and liposome.

SUMMARY OF THE INVENTION

The present invention relates to methods and compositions for lipidmatrix-assisted chemical ligation and synthesis of membranepolypeptides, compositions produced by the methods, and assays thatemploy them. The methods involve contacting a lipid matrix-incorporatedmembrane polypeptide with a ligation label comprising one or more aminoacids, where the polypeptide and label comprise amino acids havingunprotected reactive groups capable of chemoselective chemical ligation.A variety of chemoselective chemistries can be used for ligation such asnative chemical ligation, oxime-forming ligation, thioester formingligation, thioether forming ligation, hydrazone forming ligation,thiazolidine forming ligation, and oxazolidine forming ligation.Compositions of the invention include totally synthetic andsemi-synthetic lipid matrix-embedded membrane polypeptides that areproduced by the lipid matrix-assisted chemical ligation method of theinvention.

The present invention further includes a method of forming a lipidmatrix-embedded membrane polypeptide comprising a ligation site amenableto chemoselective chemical ligation when treated with a reagent thatcleaves the polypeptide directly adjacent to a residue amenable tochemoselective ligation. This aspect of the invention involvescontacting a membrane polypeptide that is embedded in a lipid matrixwith a reagent that selectively cleaves the polypeptide at a specificsite so as to generate a lipid matrix-embedded membrane polypeptide withan unprotected N-terminal or C-terminal residue that is amenable tochemoselective chemical ligation. The cleavage site may occur naturallyin the polypeptide or the polypeptide can be engineered to contain oneor more such sites.

The present invention also includes a method of detecting a ligand thatdirectly or indirectly interacts with a folded membrane polypeptideembedded in a lipid matrix. This aspect of the invention involvescontacting with a ligand, a lipid matrix-embedded synthetic orsemi-synthetic membrane polypeptide produced by lipid matrix-assistedchemical ligation, where the ligand and/or the membrane polypeptidecomprise a detectable label. The ligands may be derived from any numberof sources including naturally occurring ligands and synthetic andsemi-synthetic sources, such compound libraries. This method isparticularly useful for diagnostic assays, screening new compounds fordrug development, and other structural and functional assays that employbinding of a ligand to a prefolded membrane polypeptide.

Also provided is support matrix suitable for screening assays, where thesupport matrix comprises a detectably labeled lipid matrix-embeddedmembrane polypeptide attached thereto through a chemical handle. Thepresent invention also provides kits having at least one or morecompositions of the invention.

The present invention further provides a method for on-resin labeling apeptide with a chelator-sensitized metal ion probe. The method involveslabeling one or more amino acids of a peptide attached to a resin with azwitterionic chelator moiety label capable of chelating metal ions. Alsoincluded is a method to increase the solubility of a zwitterionicchelating agent. This method involves combining an insolublezwitterionic chelating agent with a solubilizing agent that produces asoluble salt form of the zwitterionic chelating agent. The inventionalso provides s a composition comprising a soluble salt form of azwitterionic chelator agent.

The methods and compositions of the invention permit unprecedentedaccess to membrane polypeptides and their site-specific labeling withone or more detectable labels. The methods and compositions also havemultiple additional uses. For example, they can be used to assay ligandbinding to membrane polypeptides and domains comprising a receptor, andthus are extremely useful for structure/function studies, drugscreening/selection/design, and diagnostics and the like, includinghigh-throughput applications. The methods and compositions of theinvention are particularly suited for FRET analyses of previouslyinaccessible membrane polypeptides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D illustrates various ways membrane polypeptides associate witha lipid bilayer.

FIG. 2 illustrates various chemical ligation schemes for lipidmatrix-embedded membrane polypeptides.

FIGS. 3A-C shows schematic demonstrating site-specific protease cleavageand native chemical ligation of a lipid matrix-embedded membranepolypeptide.

FIG. 4 shows a typical matrix assisted laser desorption (MALDI) massspectra output that monitors creation of a free unprotected N-terminalcysteine residue by site-specific cleavage, followed by native chemicalligation of a ligation label of choice to this residue.

FIG. 5 illustrates donor-acceptor chromophore pair labeling schemes forresonance energy transfer (FRET) assays of a lipid-matrix embeddedmembrane polypeptide.

FIG. 6 illustrates FRET assays for ion channels.

FIG. 7 illustrates FRET proximity assays for ion channels.

FIG. 8 illustrates structure-activity relationship (SAR) assays by NMRfor ion channels.

FIG. 9 illustrates reversed-phase HPLC and mass analysis employingelectrospray mass spectrometry of HIV vpu ion channel precursorN-terminal and C-terminal peptides at time=0 hours and ligation productat time=3 hours following initiation of ligation indodecylphosphocholine (DPC) micelle.

FIG. 10 illustrates gel permeation HPLC and mass analysis employingMALDI mass spectrometry of S. livdans potassium ion channel (KCSA)precursor N-terminal and C-terminal peptides and ligation productfollowing initiation of and overnight ligation in1-monooleoyl-racglycerol (C18:1, {cis}-9) (MO) cubic lipidic phase(CLP).

DEFINITIONS

Amino Acids: Include the 20 genetically coded amino acids, rare orunusual amino acids that are found in nature, and any of thenon-naturally occurring and modified amino acids. Sometimes referred toas amino acid residues when in the context of a peptide, polypeptide orprotein.

Chemoselective chemical ligation: Chemically selective reactioninvolving covalent ligation of (1) a first unprotected amino acid,peptide or polypeptide with (2) a second amino acid, peptide orpolypeptide. Any chemoselective reaction chemistry that can be appliedto ligation of unprotected peptide segments.

Chromophore: Chemical moiety that displays light absorption within theultraviolet (250-400 nm) to visible (400-700 nm) light regions of thespectrum. Includes fluorophores, dyes and donor and acceptor moieties ofa resonance energy transfer system. Can occur naturally (intrinsic) orbe added (extrinsic) to a biological molecule such as a peptide,polypeptide, carbohydrate or lipid.

Cleavage Site: Amino acid sequence capable of being cleaved by a reagentcomprising a chemical or protease that facilitates hydrolysis of apeptide bond between two amino acids of a target polypeptide.

Hydrazone chemical ligation: Chemoselective reaction involving ligationof a first unprotected amino acid, peptide or polypeptide having ahydrazine moiety and a second unprotected amino acid, peptide orpolypeptide having an aldehyde or ketone moiety resulting in theformation of a ligation product containing a hydrazone moiety at theligation site. The backbone structure of a peptide or polypeptideproduct resulting form hydrazone forming chemical ligation isdistinguishable from that of a peptide or polypeptide occurring innature or via recombinant expression.

Ligation Label: A chemical moiety comprising one or more amino acids.Can be a peptide or polypeptide.

Lipid Matrix: A molecular matrix containing natural, synthetic orcombinations thereof of lipid molecules capable of forming a lyotropicphase (i.e., formation of an ordered structure upon interaction withwater). Also referred to as a lipid membrane. The lipid molecules haveintermediate molecular weight of about 100-5000 and contain asubstantial portion of aliphatic or aromatic hydrocarbon. Includes oneor more polar lipids such as phospholipids, lysophospholipids,sphingolipids, and glycolipids capable of forming lamellar bilayers andother lipid aggregates having various two-dimensional lamellae and/orhexagonal phase lattice structures and/or three-dimensional cubic phaselattice structures.

Membrane Polypeptide: A polypeptide comprising a hydrophobic moiety thatanchors the polypeptide to the lipid membrane. Also referred to as amembrane peptide or membrane protein. Can include one or more lipidmembrane anchoring domains, such as a hydrophobic segment of thepolypeptide, a covalently attached fatty acid chain or a covalentlyattached lipid chain. May be a single or multi-pass-transmembranepolypeptide. May include one or more extramembranous amino acid residuesthat preferentially interact with the aqueous phase, such as residuescomprising an extracellular or intracellular loop.

Native chemical ligation: Chemoselective reaction involving ligation ofa first unprotected amino acid, peptide or polypeptide and a secondunprotected amino acid, peptide or polypeptide resulting in theformation of an amide bond having a backbone structure indistinguishablefrom that of a peptide or polypeptide occurring in nature or viarecombinant expression.

Oxazolidine chemical ligation: Chemoselective reaction involvingligation of a first unprotected amino acid, peptide or polypeptidehaving an aldehyde or ketone moiety and a second unprotected amino acid,peptide or polypeptide having a 1-amino, 2-ol moiety resulting in theformation of an oxazolidine moiety at the ligation site. The backbonestructure of a peptide or polypeptide product resulting from oxazolidineforming chemical ligation is distinguishable from that of a peptide orpolypeptide occurring in nature or via recombinant expression.

Oxime chemical ligation: Chemoselective reaction involving ligation of afirst unprotected amino acid, peptide or polypeptide having an amino-oxymoiety and a second unprotected amino acid, peptide or polypeptidehaving an aldehyde or ketone moiety resulting in the formation of anoxime moiety at the ligation site. The backbone structure of a peptideor polypeptide product resulting from oxime chemical ligation isdistinguishable from that of a peptide or polypeptide occurring innature or via recombinant expression.

Peptide: A polymer of at least two monomers, wherein the monomers areamino acids, sometimes referred to as amino acid residues, which arejoined together via an amide bond. May have either a completely nativeamide backbone or an unnatural backbone or a mixture thereof. Can beprepared by known synthetic methods, including solution synthesis,stepwise solid phase synthesis, segment condensation, and convergentcondensation. Can be synthesized ribosomally in cell or in a cell freesystem, or generated by proteolysis of larger polypeptide segments. Canbe synthesized by a combination of chemical and ribosomal methods.

Polypeptide: A polymer comprising three or more monomers, wherein themonomers are amino acids, sometimes referred to as amino acid residues,which are joined together via an amide bond. Also referred to as apeptide or protein. Can comprise native amide bonds or any of the knownunnatural peptide backbones or a mixture thereof. Range in size from 3to 1000 amino acid residues, preferably from 3-100 amino acid residues,more preferably from 10-60 amino acid residues, and most preferably from20-50 amino acid residues. Segments or all of the polypeptide can beprepared by known synthetic methods, including solution synthesis,stepwise solid phase synthesis, segment condensation, and convergentcondensation. Segments or all of the polypeptide also can be preparedribosomally in a cell or in a cell-free translation system, or generatedby proteolysis of larger polypeptide segments. Can be synthesized by acombination of chemical and ribosomal methods.

Thiazolidine chemical ligation: Chemoselective reaction involvingligation of a first unprotected amino acid, peptide or polypeptidehaving an aldehyde or ketone moiety and a second unprotected amino acid,peptide or polypeptide having a 1-amino, 2-thiol moiety resulting in theformation of a thiazolidine moiety at the ligation site. The backbonestructure of a peptide or polypeptide product resulting fromthiazolidine chemical ligation is distinguishable from that of a peptideor polypeptide occurring in nature or via recombinant expression.

Thioester chemical ligation: Chemoselective reaction involving ligationof a first unprotected amino acid, peptide or polypeptide and a secondunprotected amino acid, peptide or polypeptide resulting in theformation of a thioester bond at the ligation site. The backbonestructure of a peptide or polypeptide product resulting from thioesterchemical ligation is distinguishable from that of a peptide orpolypeptide occurring in nature or via recombinant expression.

Thioether chemical ligation: Chemoselective reaction involving ligationof a first unprotected amino acid, peptide or polypeptide and a secondunprotected amino acid, peptide or polypeptide resulting in theformation of a thioether bond at the ligation site. The backbonestructure of a peptide or polypeptide product resulting from thioetherchemical ligation is distinguishable from that of a peptide orpolypeptide occurring in nature or via recombinant expression.

DESCRIPTION OF SPECIFIC EMBODIMENTS

The present invention relates to methods and compositions for lipidmatrix-assisted chemical ligation and synthesis of membrane polypeptidesor membrane polypeptide domains that are incorporated into a lipidmatrix. The present invention also relates to compositions produced bythe methods of the invention and assays that employ them.

Lipid matrix-assisted chemical ligation and synthesis methods of theinvention involve chemoselective ligation of a ligation label to amembrane polypeptide embedded in a lipid matrix. The membranepolypeptide and ligation label components possess unprotected reactivegroups that selectively react to yield a covalent bond at the ligationsite, also referred to as a chemoselective ligation site. The lipidmatrix is exploited to provide (1) environment for maintaining themembrane polypeptide in a prefolded state, and (2) positioning ofreactive groups for chemoselective ligation. This aspect of theinvention is exemplified in Scheme 1.

“X_(n)” represents one or more amino acids. “R^(1” and “R) ²” representunprotected reactive groups capable of chemoselective chemical ligationwith respect to each other. “MP” represents a membrane polypeptidecomprising at least one hydrophobic (non-polar) region embedded in alipid matrix. Dashes “—” connecting groups depict covalent bonds.Chemoselective interaction between the unprotected reactive groups ofligation components “Xn-R₂” and “R₁-MP” yields the chemical ligationproduct “Xn-R₃-MP,” where “R₃” represents resultant covalent linkageformed by chemoselective chemical ligation between Xn-R₂ and R₁-MP.Reaction intermediates and side-products are not depicted. A totallysynthetic product is produced where all ligation components are man-madeby chemical synthesis, i.e., ribosomal-free synthesis. A semi-syntheticproduct is produced where at least part of a ligation component is madeby biological synthesis, i.e., ribosomally in a cell or cell-freetranslation system, and another part is made by chemical synthesis.

A variety of chemistries can be employed in accordance with the methodsof the invention. Any chemoselective reaction chemistry that can beapplied to the ligation of unprotected peptide segments is amenable tolipid matrix-assisted chemoselective chemical ligation. Thesechemistries include, but are not limited to, native chemical ligation(Dawson, et al., Science (1994) 266:776-779; Kent, et al., WO 96/34878),extended general chemical ligation (Kent, et al., WO 98/28434),oxime-forming chemical ligation (Rose, et al., J. Amer. Chem. Soc.(1994) 116:30-33), thioester forming ligation (Schnölzer, et al.,Science (1992) 256:221-225), thioether forming ligation (Englebretsen,et al., Tet. Letts. (1995) 36(48):8871-8874), hydrazone forming ligation(Gaertner, et al., Bioconj. Chem. (1994) 5(4):333-338), thiazolidineforming ligation and oxazolidine forming ligation (Zhang, et al., Proc.Natl. Acad. Sci. (1998) 95(16):9184-9189; Tam, et al., WO 95/00846).

Reaction conditions for a given ligation chemistry are selected tomaintain the desired interaction of the lipid matrix-embedded membranepolypeptide and ligation label components. For example, pH andtemperature, water-solubility of the ligation label, ratio of lipid topolypeptide and label, water content and composition of the lipid matrixcan be varied to optimize ligation. Addition or exclusion of reagentsthat solubilize the lipid matrix and/or the membrane polypeptide todifferent extents may further be used to control the specificity andrate of the desired ligation reaction, i.e., control exposure andpresentation of reactive groups by manipulating solubility of the lipidmatrix and/or membrane polypeptide. Reaction conditions are readilydetermined by assaying for the desired chemoselective reaction productcompared to one or more internal and/or external controls.

For lipid matrix-assisted native chemical ligation, the membranepolypeptide and ligation-label comprise a compatible native chemicalligation component pairing in which one of the components provides acysteine having an unprotected amino group and the other componentprovides an amino acid having an unprotected α-thioester group. Thesegroups are capable of chemically reacting to yield a native peptide bondat the ligation site.

The membrane polypeptide and ligation label for lipid matrix-assistedoxime-forming chemical ligation comprise a compatible oxime-formingchemical ligation component pairing in which one of the componentsprovides an unprotected amino acid having an aldehyde or ketone moietyand the other component provides an unprotected amino acid having anamino-oxy moiety. These groups are capable of chemically reacting toyield a ligation product having an oxime moiety at the ligation site.

For lipid matrix-assisted thioester-forming chemical ligation, themembrane polypeptide and ligation label comprise a compatiblethioester-forming chemical ligation component pairing in which one ofthe components provides an unprotected amino acid having a haloacetylmoiety and the other component provides an unprotected amino acid havingan α-thiocarboxylate moiety. These groups are capable of chemicallyreacting to yield a ligation product having an thioester moiety at theligation site.

The membrane polypeptide and ligation label for lipid matrix-assistedthioether-forming chemical ligation comprise a compatiblethioether-forming chemical ligation component pairing in which one ofthe components provides an unprotected amino acid having a haloacetylmoiety and the other component provides an unprotected amino acid havingan alkyl thiol moiety. These groups are capable of chemically reactingto yield a ligation product having a thioether moiety at the ligationsite.

The membrane polypeptide and ligation label for lipid matrix-assistedhydrazone-forming chemical ligation comprise a compatiblehydrazone-forming chemical ligation component pairing in which one ofthe components provides an unprotected amino acid having an aldehyde orketone moiety and the other component provides an unprotected amino acidhaving an hydrazine moiety. These groups are capable of chemicallyreacting to yield a ligation product having a hydrazone moiety at theligation site.

For lipid matrix-assisted thiazolidine-forming chemical ligation, themembrane polypeptide and ligation label comprise a compatiblethiazolidine-forming chemical ligation component pairing in which one ofthe components provides an unprotected amino acid having a 1-amino,2-thiol moiety and the other component provides an unprotected aminoacid having an aldehyde or a ketone moiety. These groups are capable ofchemically reacting to yield a ligation product having a thiazolidinemoiety at the ligation site.

For lipid matrix-assisted oxazolidine-forming chemical ligation, themembrane polypeptide and ligation label comprise a compatibleoxazolidine-forming chemical ligation component pairing in which one ofthe components provides an unprotected amino acid having a 1-amino,2-hydroxyl moiety and the other component provides an unprotected aminoacid presenting an aldehyde or a ketone moiety. These groups are capableof chemically reacting to yield a ligation product having an oxazolidinemoiety at the ligation site.

As is readily apparent, any combination of ligation components that areadapted for chemoselective chemical ligation to yield a lipidmatrix-embedded polypeptide product having a covalent bond at theligation site are considered part of the invention, provided that atleast one of the reaction ligation components comprises a membranepolypeptide embedded in a lipid matrix. In particular, the membranepolypeptide ligation component is required to comprise at least onehydrophobic region that anchors the polypeptide to a lipid matrix. Anexample of an anchoring region is a cell membrane anchor comprisinghydrophobic α-helix and/or β-barrel transmembrane components. Themembrane polypeptide also may include native amide bonds or any of theknown unnatural peptide backbones or a mixture thereof, and otherchemical differences from a native amino acid sequence, such as anunnatural amino acid comprising a chromophore or other detectable moietycompatible with maintenance of the membrane polypeptide in the lipidmatrix. This includes single-pass and multi-pass transmembranepolypeptides or domains thereof having a native or non-native sequenceof amino acids that embed in and interact with a hydrophobic portion ofa lipid matrix, and may include one or more extramembranous amino acidsthat protrude into aqueous phase environment formed by the lipidmatrix-water interface. Examples of extramembranous amino acids includethose that are part of or form an intracellular or extracellular loopconnecting two membrane anchors, an N- or C-terminal sequence, asequence necessary to facilitate insertion of the polypeptide into alipid membrane, and/or some other linker or capping sequence ofinterest.

The membrane polypeptide ligation component preferably comprises anamino acid sequence that permits both insertion and anchoring in a lipidmembrane. For example, the membrane polypeptide generally comprises anamino acid sequence capable of forming a transmembrane structure, suchas an amphipathic α-helix, organized such that non-polar residues are incontact with the membrane interior and charged or polar residues are incontact with the aqueous phase. Membrane polypeptide ligation componentsof this type are of sufficient length to span the lipid bilayer, andthus are typically greater than 15-20 amino acids in length. They mayrange in size up to the full length of a naturally occurring membranepolypeptide, and may include one or more amino acids in addition to suchfull-length polypeptides, provided the membrane polypeptide is capableof incorporation in the lipid matrix.

The lipid matrix into which the membrane polypeptide component isincorporated includes natural and synthetic lipids capable of forming alyotropic phase (i.e., formation of an ordered membrane type structureon interaction with water, such as a liquid crystalline phase). Theseinclude polar lipids such as phospholipids, lysophospholipids,sphingolipids, and glycolipids capable of forming lamellar bilayers andother lipid aggregates. Preferred lipid matrices form stable membranemonolayers or bilayers and aggregate phases thereof. Of particularinterest are lipids that form stable cubic phases (cubic lipidic phaseor CLP matrix) (Luzatti, et al., Nature (1968) 218:1031-1034; andLindblom, et al., Biochimica et Biophysica Acta (1989) 988:221-256). Acubic phase is one in which the lipid aggregates form athree-dimensional lattice. The lipid aggregate units can have differentshapes such as spheres, rods, or lamellae. In contrast, lamellar liquidcrystalline phases exhibit a one-dimensional periodicity in whichlamellar units of infinite expression are stacked regularly, andhexagonal liquid crystalline phases exhibit a two-dimensionalperiodicity with rod-like aggregates of infinite length packed into ahexagonal lattice. Thus, cubic phases are optically isotropic whereaslamellar and hexagonal phases are optically anisotropic.

Lipids that form cubic phases consist of a multiple lipid bilayersprotruded by multiple aqueous channels formed between certain lipids andpolar solvents at specific lipid:solvent ratios (Luzatti, et al., supra;and Lindblom, et al., supra). The lipid diffusion rate inside the CLP iscomparable to the rate in lamellar lipid phases and that the diffusionrate in the aqueous compartments is approximately three times slowerthan in bulk water (Lindblom, et al., supra). Additionally, membranepolypeptides can exhibit full activity when incorporated into cubicphase lipids (Portmann, et al., J. Phys. Chem. (1991) 95:8437-8440).Since CLPs are optically isotropic, they can be employed in sensitivespectrophotometric assays. Thus, manipulation of a membrane polypeptideincorporated into a lipid matrix not only provides a prefolded membranepolypeptide serving as a template for chemoselective chemical ligation,the lipid matrix itself can be exploited to facilitate assays thatrequire a lipid-mediated folded membrane polypeptide.

The ligation label component comprises one or more amino acids, and ispreferably a peptide or polypeptide. At least one amino acid of theligation label provides a reactive group that is capable of reactingwith and forming a covalent bond with a compatible reactive groupprovided by the lipid matrix-embedded membrane polypeptide. The ligationlabel also can comprise a membrane polypeptide component, for example,when modular ligation and synthesis of a multi-pass transmembranepolypeptide is desired. The ligation label also may comprise nativeand/or unnatural peptide backbone structure or unnatural amino acidresidues or other chemical differences from a native peptide sequence,such as an unnatural amino acid comprising a chromophore or otherdetectable moiety compatible with chemical ligation of the ligationlabel and the target lipid matrix-embedded membrane polypeptide.

The lipid matrix, the lipid matrix-embedded membrane polypeptide, and/orligation label of the invention also may employ one or more chemicaltags. The chemical tag may be utilized for multiple purposes such aspart of the synthesis process, purification, anchoring to a supportmatrix, detection and the like. Of particular interest is a chemical tagprovided by an unnatural amino acid comprising a chromophore. Thisincludes a chromophore that is an acceptor and/or donor moiety of anacceptor-donor resonance energy transfer pair. Of particular interestare synthesis and purification handles, as well as detectable labels andoptionally chemical moieties for attaching the lipid matrix or the lipidmatrix embedded membrane polypeptide to a support matrix for screeningand diagnostic assays and the like. Various labels described herein aresuitable for this purpose. As can be appreciated, in some instances itmay be advantageous to utilize a given chemical tag for more than onepurpose, e.g., both as a handle for attaching to support matrix and as adetectable label. Examples of chemical tags include metal binding tags(e.g., his-tags), carbohydrate/substrate binding tags (e.g., celluloseand chitin binding domains), antibodies and antibody fragment tags,isotopic labels, haptens such as biotin and various unnatural aminoacids comprising a chromophore. A chemical tag also may include acleavable linker.

Exploiting the lipid matrix to perform chemical ligation of a membranepolypeptide embedded in a lipid matrix provides unprecedentedsite-specific ligation, detectable labeling and modular synthesis ofprefolded membrane polypeptides. Not only does the lipid matrix providethe proper environment for insertion, folding and presentation of achemical ligation site, embedding the membrane polypeptide component ina lipid matrix exploits the lipid matrix itself as a novel component forcontrolling specificity of the chemical ligation reaction. For example,a ligation site on the membrane polypeptide can be selected such that itis buried within a lipid membrane bilayer of the matrix. In thissituation chemical ligation of a hydrophobic ligation label will bepreferred compared to a hydrophilic ligation label. Alternatively, aligation site can be extra-membranous such that ligation of ahydrophilic ligation label is preferred. Another variation made possibleby exploiting the lipid matrix component in combination with thelocation of the ligation site is construction of a lipid membrane systemdesigned to partition two aqueous phases from each other, such as withclosed membrane vesicles (e.g., liposomes), where a ligation site isselected for exposure on one side of the membrane. Exposure of aligation site can be further adjusted to control the rate and/or extentof the ligation reaction by the addition or exclusion of reagents thatsolubilize the lipid matrix to different extents. Furthermore, theextent of exposure and reactivity of the ligation site can be controlledby adjusting the reaction conditions such as pH and temperature, as wellas the water-solubility of the ligation peptide, water content andcomposition of the lipid matrix. Thus, depending on the position andorientation of the ligation site with respect to the lipid membrane inwhich it is embedded, the solubility of the ligation label, and theintegrity and composition of the lipid matrix, the specificity and rateof the ligation reaction can be controlled.

The present invention further includes a method of forming achemoselective ligation site in a lipid matrix-embedded membranepolypeptide when treated with a cleavage reagent that selectivelycleaves the polypeptide directly adjacent to an amino acid residue thatprovides a reactive group for chemical ligation. This aspect of theinvention involves contacting a membrane polypeptide that is embedded ina lipid matrix with a reagent that selectively cleaves the polypeptideat a specific site so as to generate a polypeptide comprising anN-terminal residue having an unprotected amino group or a C-terminalresidue having an unprotected carboxyl group. This aspect of theinvention is exemplified in Scheme 2.

“Y_(n)” represents an amino acid sequence comprising a cleavage site,such as a chemical or protease cleavage site, that permits cleavagedirectly adjacent to the amino acid residue providing the R₁ reactivegroup for subsequent chemoselective chemical ligation.

The cleavage site may occur naturally in the polypeptide or thepolypeptide can be engineered to contain one or more such sites. One ormore cleavage sites also may be present in the ligation label if desireddepending on its intended end use. A naturally occurring or engineeredcleavage site therefore allows for the generation of a free andunprotected N-terminal and/or C-terminal residue by site-specificproteolysis. A ligation component generated in this manner thereforecontains a residue having an unprotected reactive group amenable tolipid matrix-assisted chemical ligation methods of the invention. It isreadily apparent from Scheme 2 that one or more cleavage sites can beincorporated in any combination with a target ligation component of theinvention, provided that upon cleavage the cleavage product is adaptedfor lipid matrix-assisted chemoselective chemical ligation.

The cleavage site can be a protease or a chemical cleavage site. Acleavage site is chosen and/or incorporated into the polypeptide duringits synthesis so that upon exposure to the complementary cleavagereagent, the polypeptide is selectively cleaved at the site so as togenerate a polypeptide having a free, unprotected amino acid residuehaving a chemoselective reactive group that is compatible for ligationwith a complementary reactive group provided by the ligation label. Byway of example, when native chemical ligation chemistry is employed andan endopeptidase cleavage site sequence is incorporated between a linkeror capping sequence and a Cys-polypeptide, the cleavage site permitsremoval of the linker or capping sequence and generation of the desiredunprotected Cys-polypeptide. Alternatively, the cleavage site may bepositioned adjacent to a residue capable of spontaneous ligation with acomplementary reactive group donated by a properly positionedneighboring residue.

Some commonly encountered protease cleavage sites are: Thrombin(KeyValProArg/ GlySer) (SEQ ID NO: 31); Factor Xa Protease(IleGluGlyArg) (SEQ ID NO: 32); Enterokinase (AspAspAspAspLys) (SEQ IDNO: 33); rTEV (GluAsnLeuTyrPheGlnlGly) (SEQ ID NO: 34) which is arecombinant endopeptidase from the Tobacco Etch Virus; and 3C Humanrhino virus Protease (Phannacia Biotech) (LeuGluValLeuPhe GlnlGlyPro)(SEQ ID NO: 35).

Various chemical cleavage sites also are known and include, but are notlimited to, the intein protein-splicing elements (Dalgaard, et al.,Nucleic Acids Res. (1997) 25(6):4626-4638) and cyanogen bromide cleavagesites. Inteins can be constructed which fail to splice, but insteadcleave the peptide bond at either splice junction (Xu, et al., EMBO J(1996) 15(19):5146-5153; and Chong, et al., J. Biol. Chem.(1996)271:22159-22168). For example, the intein sequence derived fromthe Saccharomyces cerevisiae VMA1 gene can be modified such that itundergoes a self-cleavage reaction at its N-terminus at low temperaturesin the presence of thiols such as 1,4-dithiothreitol (DTT),2-mercaptoethanol or cysteine (Chong, et al., Gene (1997) 192:271-281).

Cyanogen bromide (CnBr) cleaves at internal methionine (Met) residues ofa polypeptide sequence. Cleavage with CnBr yields two or more fragments,with the fragments containing C-terminal residues internal to theoriginal polypeptide sequence having an activated alpha-carboxylfunctionality, e.g. cyanogen bromide cleavage at an internal Met residueto give a fragment with a C-terminal homoserine lactone. For somepolypeptide, the fragments will re-associate under folding conditions toyield a folded polypeptide-like structure that promotes reaction betweenthe segments to give a reasonable yield (often 40-60%) of thefull-length polypeptide chain (now containing homoserine residues wherethere were Met residues subjected to cyanogen bromide cleavage)(Woods,et al., J. Biol. Chem. (1996), 271:32008-32015).

In general, a cleavage site for generating a ligation site amenable to adesired chemical ligation chemistry usually is selected to be unique,i.e., it occurs only once in the target polypeptide. However, when morethan one cleavage site is present in a target polypeptide that isrecognized and cleaved by the same cleavage reagent, if desired one ormore of such sites can be permanently or temporarily blocked from accessto the cleavage reagent and/or removed during synthesis. In particular,positioning of the cleavage site may be controlled from exposure to thecleavage reagent by exploiting its position in the lipid matrix asdescribed above for the chemoselective ligation site. Cleavage sites canbe removed during synthesis of the membrane polypeptide by replacing,inserting or deleting one or more residues of the cleavage reagentrecognition sequence, and/or incorporating one or more unnatural aminoacids that achieve the same result. A cleavage site also may be blockedby agents that bind to the membrane polypeptide, including ligands thatbind the polypeptide and remove accessibility to all or part of thecleavage site. However a cleavage site is blocked or removed, one ofordinary skill in the art will recognize that the method is selectedsuch that upon cleavage the membrane polypeptide is capable ofchemoselective chemical ligation to a target ligation component ofinterest.

Inclusion of a cleavage site for generating a chemoselective ligationsite is particularly advantageous when the membrane polypeptide isadapted with a capping, signal or other type of leader sequencecontaining information necessary to facilitate or improve efficacy ofinsertion into a lipid membrane, insertion into particular membranes ofchoice, detection, and/or purification. For example, cleaving a membranepolypeptide after it is embedded within a lipid membrane matrix permitsselection of properly folded polypeptides, as well as removal ofsequences that may be needed to initiate proper insertion and folding,but are no longer needed once a hydrophobic anchoring sequence isembedded in the lipid matrix. The cleavage site also can be used fordetection and/or purification of properly incorporated and foldedpolypeptides. For example, when a membrane polypeptide is selected tocomprise a cleavage site designed for extramembranous exposure, thenpolypeptides having the appropriate configuration in a lipid membrane,and thus extramembranous exposure of the cleavage site, are likely toexhibit maximum sensitivity to cleavage when a water-soluble cleavagereagent is employed under aqueous conditions, such as a water-solubleendoprotease. A cleavage site for generating a chemoselective ligationsite may also be included when a purification handle or tag is includedas part of a fusion polypeptide to ease its purification followingsynthesis. Thus, inclusion of a cleavage site designed to generate achemoselective ligation site can be exploited for multiple purposes.

The membrane polypeptide and ligation label component pairings suitablefor the chemical ligation approaches exemplified by Schemes 1-2 can bedesigned de novo or derived from virtually any known membrane proteinsystem, including those derived from viral, eukaryotic, prokaryotic, andarchaebacterial systems, including psychrophilic, mesophilic andthermophilic organisms, and particularly the two major classes ofmembrane proteins, i.e., those that insert α-helices into the lipidbilayer, and those that form pores by a β-barrel strands. Examplesinclude membrane associated receptors, transporter proteins (e.g., ionand other channels such as potassium, sodium, proton, chloride channels,pores; active transporters such as TEXAN drug transporters, mini-TEXANSand ABC drug transporters and antiporters such as the H+/glucoseantiporter); enzymes (e.g. leucotriene C4 synthase); and immunogens(e.g. tumor metastasis-associated antigen). Of particular interest areenzyme-linked receptors, fibronectin-like receptors, the seventransmembrane receptors, and the ion channel receptors, including thetyrosine and serine-threonine kinases, and guanylate cyclase families ofenzyme-linked receptors. Examples of the tyrosine kinase family ofreceptors include epidermal growth factor, insulin, platelet-derivedgrowth factor, and nerve growth factor. Examples of the serine kinasefamily of receptors include growth factor β-family. Examples of theguanylate cyclase family includes those receptors that generate cyclicGMP (cGMP) in response to atrial natriuretic factors. Examples of theseven-transmembrane receptors include those membrane proteins that bindcatecholamines, histamines, prostaglandins, etc., and the opsins,vasopressin, chemokine and melanocortin receptors. Examples of the ionchannel receptors are represented by the ligand- and voltage-gatedchannel membrane protein receptors, and include the acetylcholineactivated sodium channels, glycine and gamma-aminoisobutyric acidactivated chloride channels, and serotonin and glutamate activatedcalcium channels, and the family of cyclic nucleotide-gated channels(cAMP and cGMP), and the family of inositol 1,4,5-triphosphate (IP3) andthe cyclic ADP-ribose receptors that modulate calcium storage. One ofordinary skill in the art will recognize that nucleic acid and/or aminoacid sequences for the above and additional membrane polypeptides can beidentified in various genomic and protein related databases. Examples ofpublicly accessible databases include as GenBank (Benson, et al.,Nucleic Acids Res (1998)26(1):1-7; USA National Center for BiotechnologyInformation, National Library of Medicine, National Institutes ofHealth, Bethesda, Md., USA), TIGR Database (The Institute for GenomicResearch, Rockville, Md., USA), Protein Data Bank (Brookhaven NationalLaboratory, USA), and the ExPASy and Swiss-Protein database (SwissInstitute of Bioinformatics, Geneve, Switzerland).

In selecting the membrane polypeptide and ligation label componentpairings and their respective ligation sites, complementarychemoselective pairings can be identified by analysis of a targetpolypeptide's structure in the context of its function using one or moreroutine procedures such as biological, thermodynamic, computational andstructural techniques known in the art and as described herein. Theinformation is used to select one or more chemoselective ligation sitespresent in the native structure and/or engineered into a synthetic formof the molecule. In particular, structural and functional informationcan be obtained using standard techniques including homology comparisonsto other polypeptides having similar amino acid sequences and domains,preferably other polypeptides for which at least some structural andfunctional information is known. For an exemplary list of membranepolypeptides of known three-dimensional structure, see Preusch, et al.,Nature Struct. Biol. (1998) 5:12-14.

Uniqueness of the ligation site, stability of the ligation components,specificity and completeness of the ligation reaction, and stability andfunction of the lipid matrix-embedded membrane polypeptide ligationproduct are considerations of the selection process. In particular,ligation component pairings are preferably designed to maximizeselectivity of the ligation reaction and stability of the ligationproduct within the lipid matrix. This includes design of linker orcapping sequences comprising one or more cleavage sites employed forincorporation and generation of a chemoselective ligation site of aligation component in the lipid matrix. For example, mutagenesis,thermodynamic, computational, modeling and/or any technique that revealsfunctional and/or structural information regarding a target polypeptideof interest can be used for this process. These techniques includeimmunological and chromatographic analyses, fluorescence resonanceenergy transfer (FRET), circular dichroism (CD), nuclear magneticresonance (NRM), electron and x-ray crystallography, electronmicroscopy, Raman laser spectroscopy and the like, which are commonlyexploited for designing and characterizing membrane polypeptide systems.(See, e.g., Newman, R., Methods Mol. Biol. (1996) 56:365-387; Muller, etal., J. Struct. Biol. (1997) 119(2):149-157; Fleming, et al., J. MolBiol. (1997) 272:266-27; Haltia, et al., Biochemistry (1994) 33(32):9731-9740.5; Swords, et al., Biochem J. (1993) 289(1): 215-219; Wallin,et al., Protein Sci. (1997) 6(4):808-815; Goormaghtigh et al., SubcellBiochem. (1994) 23:405-450). Muller, et al., Biophys J. (1996)70(4):1796-1802; Sami, et al., Biochimica et Biophysica Acta (1992)1105(1):148-154. Wang, et al., J. Mol. Biol. (1994) 237(1):1-4; Watts,et al., Mol. Memb. Biol. (1995) 12(3):233-246; Bloom, M., Biophys J.(1995) 69(5):1631-1632; and Gutierrez-Merino, et al., Biochem Soc.Trans. (1994) 22(3):784-788).

When structural information is not immediately available, thechemoselective chemical ligation components can be identified andmodeled in two-dimensions by various techniques known in the art. Forexample, amphipathic α-helix segments that span a lipid membranebilayer, and thus greater than about 15-20 residues, can be identifiedin the primary structure using secondary structure predictionalgorithms. Segments greater than 15-20 residues are selected based onsequence similarity between members of a superfamily, and then one ormore ligation sites are identified for compatibility with a ligationlabel that may or may not be part of the original two-dimensional model.(See, e.g., Reithmeier, R. A., Curr. Opin. Struct. Biol. (1995)5(4):491-500; Du, et al., Protein Eng. (1994) 7(10):1221-1229). Apreferred technique is homology modeling and database alignments withthe aid of one or more computer programs suited for membrane polypeptidemodeling and/or prediction. For example, the program “TmPred” andTopPredII” can be used to make predictions of membrane-spanning regionsand their orientation, which is based on the statistical analysis of adatabase of transmembrane proteins present in the SwissProt database.(Gunnar von Heijne, J. Mol. Biol. (1992) 225:487-494; Hoppe-Seyler,Biol. Chem. (1993) 347:166; and Claros, et al., Comput Appl Biosci.(1994) 10(6):685-686). Other programs can be used and include: “DAS”(Cserzo, et al., Prot. Eng. (1997) 10(6):673-676); “PHDhtm” (Rost, etal., Protein Science (1995) 4:521-533); and “SOSUI” (Mitaku Laboratory,Department of Biotechnology, Tokyo University of Agriculture andTechnology).

The ligation components and complementary pairings thereof also can beselected by modeling them in three-dimensions at the atomic level tosimulate the ligated product and/or the pre-ligation reactioncomponents. First, a sequence alignment between the polypeptide to bemodeled and a polypeptide of known structure is established. Second, abackbone structure is generated based on this alignment. This isnormally the backbone of the most homologous structure, but a hybridbackbone also may be used. Third, side chains are then placed in themodel. Various techniques like Monte Carlo procedures, tree searchingalgorithms etc., can be used to model rotomer side chains havingmultiple possible conformations. If the polypeptide to be modeled hasinsertions or deletions with respect to the known structure, loops arere-modeled, or modeled ab initio. Database searches for loops withsimilar anchoring points in the structure are often used to build theseloops, but energy based ab initio modeling techniques also can beemployed. Energy minimizations, sometimes combined with moleculardynamics, are then normally used for optimization of the finalstructure. The quality of the model is then assessed, including visualinspection, to verify that the structural aspects of the model are notcontradicting what is known about the functional aspects of themolecule.

The three-dimensional models are preferably generated using a computerprogram that is suitable for modeling membrane polypeptides. (Vriend,G., “Molecular Modeling of GPCRs,” In: 7TM (1995) vol. 5). Examples ofcomputer programs suitable for this purpose include: “What If” (Vriend,G., J. Mol. Graph. (1990) 8:52-56; available from EMBL, Meyerhofstrasse1, 69117 Heidelberg, Germany) and “Swiss-Model” (Peitsch M C and HerzykP (1996) “Molecular modeling of G-protein coupled receptors.” In: GProtein-coupled Receptors. New opportunities for commercial development,6:6.29-6.37, N Mulford and L M Savage Eds., IBC Biomedical LibrarySeries; Peitsch, et al., Receptors and Channels (1996) 4:161-164;Peitsch, et al., “Large-scale comparative protein modeling,” In:Proteome research: new frontiers in functional genomics,” pp. 177-186,Wilkins M R, Williams K L, Appel R O, Hochstrasser D F, Eds., Springer,1997).

Factors that influence lipid matrix-assisted chemoselective ligation,and thus selection of a compatible ligation site, generally parallelthose known for soluble proteins (Woods, et al., J. Biol. Chem. (1996)271:32008-32015). First, secondary structure is not critically importantin that ligation sites within most α-helix and β-sheet are amenable toligation. Ligation sites at most turns also are okay, although ligationsites corresponding to the extremity of a β-turn are less preferred.Second, a ligation site is preferably positioned on the surface of thefolded polypeptide, as opposed to being buried or internal to the foldedpolypeptide at positions required to stabilize the folded form of thepolypeptide in the lipid environment.

Accordingly, selection of chemoselective ligation components andcomplementary pairings thereof also considers the thermodynamic cost ofexposing and/or transferring charged or highly polar uncharged compoundsinto the oil-like hydrocarbon interior of membranes. For instance,positioning of a ligation site, such as one designed for generation bysite-specific cleavage of a lipid matrix-embedded membrane polypeptide,is not only designed for ligation selectivity, but it also is designedso that the precursor and final ligation products are sufficientlystable in the lipid matrix. In this aspect of the invention, most aminoacid side chains of transmembrane segments must be non-polar (e.g. Ala,Val, Leu, Ile, Phe). Thus residue positions having a naturally occurringligation site residue or that are targeted for substitution or insertionof a ligation site residue by engineering are selected to maintain thefavorable hydrophobic interactions, such as hydrophobic packinginteractions found between the α-helix membrane anchors. Additionally,the very polar CONH groups (peptide bonds) of the polypeptide backboneof transmembrane segments are selected to participate in hydrogen bonds(H-bonds) in order to lower the cost of transferring them into thehydrocarbon interior (See, e.g., Roseman, A., J. Mol. Biol. (1988)201:621-625). Thus in selecting a compatible ligation site, thisH-bonding is most easily accomplished by maintaining the α-helices forwhich all peptide bonds are H-bonded internally. It also can beaccomplished by maintaining β-sheets for porin-type transmembraneproteins, provided that the β-strands form closed structures such as theβ-barrel. Since all membrane polypeptides of known three-dimensionalstructure adhere to these principles, the chemoselective chemicalligation sites can be selected and/or designed to adhere to theseprinciples when a folded transmembrane polypeptide is desired. In somecases appropriately placed positively charged lysine residues can beused to control the overall membrane orientation (Whitley, et al., Nat.Struct Biol. (1994) 1(12):858-862).

Compatible ligation component pairings and their chemoselective chemicalligation sites also can be identified by ligation site scanning. Thisinvolves a genetic approach to introduce amino acid residues amenable toa particular ligation chemistry by site-specific, nested and/or randommutagenesis into DNA in a cell or a cell free system, encoding a targetmembrane polypeptide or domain thereof of interest, followed byexpression of the mutated DNA and screening for membrane polypeptidescontaining functional, ligation compatible substitutions or insertions.Alternatively, mutated membrane polypeptides can be chemicallysynthesized, or a combination of genetic and chemical synthesis can beused. Whether recombinant DNA and/or chemical synthesis is employed, thepolypeptides subjected to ligation site scanning will preferablycomprise at least one membrane-anchoring segment capable of insertioninto a lipid membrane, such as a transmembrane spanning segment ortransmembrane polypeptide domain. Membrane polypeptides and ligationlabels comprising one or more of the functional mutations are thensynthesized to generate one or more of the chemoselective ligationcompatible pairings exemplified in Schemes 1-2. The ligation componentsare then subjected to the appropriate ligation chemistry of interest andscreened for ligated membrane polypeptides that properly insert and foldin the lipid matrix.

The ligation site scanning approach not only permits rapid and highthroughput screening of large number of-compatible ligation components,it also permits rapid screening for novel and diverse recombinant,synthetic and semi-synthetic molecules formed by new combinations ofmembrane polypeptide and ligation label components pairings. As anexample, the membrane polypeptide and ligation label components can beobtained from mixtures or libraries representing a diversity of chemicalstructures, such as different amino acid sequences as well as differentunnatural side-chain substituents. A library may contain certain sets ofpeptide or polypeptides containing a different chemical entity at aparticular position, and/or others that are randomly different. Suchcomponents can be recombinantly expressed, derived from phage displaylibraries, derived from chemical synthesis, or fragments andcombinations thereof. Libraries of modular cross-over membranepolypeptides and ligation components also may be constructed utilizingthe modular approach described in Kent, at al., WO 99/11655. In thisembodiment of the invention, lipid matrix-embedded membrane polypeptidesare generated by cross-over chemoselective chemical ligation of two ormore peptide segments comprising one or more functional protein modulesderived from different parent membrane polypeptides of the same class orfamily so as to generate one or more lipid matrix-embedded hybridmembrane polypeptides. This permits introduction of functional domainsand the like from different members of the same class or family ofmembrane polypeptides so as to generate libraries of great diversity,including ligation site information as well as structure-functioninformation. As can be appreciated, rapid screening and selection of oneor more ligation component pairings by ligation site scanning istherefore particularly suited for identifying compatible and diversecombinations of reaction components illustrated in Schemes 1-2 above.

For synthesis of the ligation components, any number of methods known inthe art can be employed. These include chemical and biological methodsor combinations thereof. For example, a nucleic acid sequence coding forpart or all of a ligation component can be expressed in a host cell, andrecovered using standard techniques and/or those described herein. Thisapproach is particularly useful when a membrane polypeptide isrecalcitrant to chemical synthesis alone. Peptide or polypeptides alsocan be produced using chemical methods to synthesize the desiredligation component in whole or in part.

For synthesis in a cell, polypeptides can be generated by standardtechniques. Cells that naturally express a target polypeptide can beemployed. Transfection and transformation of a host cell with DNAencoding a polypeptide of interest also can be used. For example, apolymerase chain reaction (PCR) based strategy may be used to clone atarget DNA sequence encoding all or part of a target membranepolypeptide of interest. (See, e.g., “PCR Cloning Protocols: FromMolecular Cloning to Genetic Engineering,” B. A. White, ed., HumanaPress, Methods in Molecular Biology, Vol. 67, 1997). For example, PCRcan be used for cloning through differential and subtractive approachesto cDNA analysis, performing and optimizing long-distance PCR, cloningunknown neighboring DNA, and using PCR to create and screen libraries.PCR also can be used to introduce site-specific and random mutationsinto DNA encoding a target membrane polypeptide and/or ligation label ofinterest. This approach is particularly suited for engineering cysteineligation sites.

For general cloning purposes, complementary and/or degenerateoligonucleotides corresponding to conserved motifs of the targetmembrane polypeptide may be designed to serve as primers in a cDNAand/or PCR reaction. Templates for primer design can be obtained fromany number of sources. For example, sequences, including expressedsequence tags (ESTs) can be obtained from various databases, such asGenBank, TIGR, ExPASy and Swiss-Protein databanks. Homology comparisonsperformed using any one of a number of alignment readily availableprograms that employ search engines to find the best primers in asequence based on various algorithms. Any number of commerciallyavailable sequence analysis packages, such as Lasergene, GeneWorks,DNASIS, Gene Jockey II, Gene Construction Kit, MacPlasmap, PlasmidARTIST, Protein Predictor, DNA/RNA Builder, and Quanta. (See, e.g.,“Sequence Data Analysis Guidebook,” Simon R. Swindell, ed., HumanaPress, 1996). The information can be used to design degenerate primers,nested/multiplex primers, site-directed mutagenesis, restriction enzymesites etc. Primers can be designed from homology information, andcomputer programs can be used for primer design as well. Examplesinclude “Primer Premier 4.0” for automatic primer selection (CloneTech,Inc.). The amplified cDNA and/or PCR fragment may be used to isolatefull-length clones by radioactive or non-radioactive labeling of theamplified fragment and screening a library.

Alternatively, membrane polypeptide DNA cloned from one source may beutilized to obtain the corresponding target membrane polypeptide DNAsequence from other sources. Specifically, a genomic and/or cDNA libraryconstructed from DNA and/or RNA prepared from a cell known or expectedto express the target membrane polypeptide may be used to transform aeukaryotic or prokaryotic host cell that is deficient in the putativegene. Transformation of a recombinant plasmid coding for the polypeptideinto a deficient host cell would be expected to provide the cell with acomplement product corresponding to the polypeptide of interest. In somecases, a host cell can be selected to express a particular phenotypeassociated with the target polypeptide and thus may be selected by thisproperty. For a review of cloning strategies which may be used, seee.g., Sambrook, et al., 1989, Molecular Cloning, A Laboratory Manual,Cold Springs Harbor Press, New York; and Ausubel, et al., 1989, CurrentProtocols in Molecular Biology, Green Publishing Associates and WileyInterscience, New York.

To express a target membrane polypeptide in a host cell the nucleotidesequence coding for the polypeptide, or a functional equivalent formodular assembly as described above, is inserted into an appropriateexpression vector, i.e., a vector which contains the necessary elementsfor the transcription and translation of the inserted coding sequence.Host cells containing the coding sequence and that express the targetgene product may be identified by standard techniques. For example,these include but are not limited to DNA-DNA or DNA-RNA hybridization;the presence or absence of “marker” gene functions; assessing the levelof transcription as measured by the expression of mRNA transcripts inthe host cell; and detection of the gene product as measured byimmunoassay or by its biological activity.

Once a clone producing the target polypeptide is identified, the clonemay be expanded and used to produce large amounts of the polypeptide,which may be purified using techniques well-known in the art including,but not limited to immunoaffinity purification, chromatographic methodsincluding high performance liquid chromatography or cation exchangechromatography, affinity chromatography based on affinity of thepolypeptide for a particular ligand, immunoaffinity purification usingantibodies and the like. For example, when expressed in a host cell,extraction and purification of membrane polypeptides can be performedfollowing standard techniques. (Ohlendieck, K., Methods Mol Biol. (1996)59:313-322). Ohlendieck, K., Methods Mol Biol. (1996) 59:293-304; JosicD, et al., Methods Enzymol. (1996), 271:113-134). Thus in anotherembodiment of the invention, nucleic acid encoding recombinant membranepolypeptides modified to contain one or more engineered ligation siteresidues designed to be accessible to chemical ligation are provided.

Some commonly used host cell systems for expression and recovery ofmembrane polypeptides include E. coli, Xenopus oocytes, baculovirus,vaccinia, and yeast, as well as many higher eukaryotes includingtransgenic cells in culture and in whole animals and plants. (See, e.g.,G. W. Gould, “Membrane Protein Expression Systems: A User's Guide,”Portland Press, 1994, Rocky S. Tuan, ed.; and “Recombinant GeneExpression Protocols,” Humana Press, 1996). For example, yeastexpression systems are well known and can be used to express and recovertarget membrane polypeptide of interest following standard protocols.(See, e.g., Nekrasova et al, Eur. J. Biochem. (1996) 238:28-37; GeneExpression Technology Methods in Enzymology 185:( 1990); MolecularBiology and Genetic Engineering of Yeasts, CRC Press, Inc. (1992);Herescovics, et al., FASEB (1993) 7:540-550; Larriba, G., Yeast (1993)9:441-463; Buckholz, R. G., Curr Opinion Biotech (1993) 4:538-542;Asenjo, et al., “An Expert System for Selection and Synthesis of ProteinPurification Processes Frontiers in Bioprocessing II,” pp. 358-379,American Chemical Society, (1992); Mackett, M, “Expression of MembraneProteins in Yeast Membrane Protein Expression Systems: A Users Guide,”pp. 177-218, Portland Press, (1995).

When chemical synthesis is employed, the peptide or polypeptides can belinear, cyclic or branched, and often composed of, but not limited to,the 20 genetically encoded L-amino acids. In vitro suppressionmutagenesis in a cell free system also can be used to introduceunnatural amino acids into the polypeptides (See, e.g., Cload, et al.,Chemistry and Biology (1996) 3:1033-1038) and Turcatti, et al., J. Bio.Chem. (1996) 271:19991-19998). Such chemical synthetic approaches alsopermit incorporation of novel or unusual chemical moieties includingD-amino acids, other unnatural amino acids, oxime, hydrazone, ether,thiazolidine, oxazolidine, ester or alkyl backbone bonds in place of thenormal amide bond, N- or C-alkyl substituents, side chain modifications,and constraints such as disulfide bridges and side chain amide or esterlinkages. See, for example, Wilkins, et al., Curr. Opin. Biotech. (1998)9(4):412-426, which reviews various chemistries for chemical synthesisof peptides and polypeptides.

For example, native chemical ligation and synthesis of polypeptideshaving a native peptide backbone structure is disclosed in Kent, et al.,WO 96/34878. See also Dawson, et al. (Science (1994) 266:77-779) andTam, et al. (Proc. Natl. Acad. Sci. USA (1995) 92:12485-12489).Unnatural peptide backbones also can be made by known methods (See,e.g., Schnolzer, et al., Science (1992) 256:221-225; Rose, et al., J.Am. Chem. Soc. (1994) 116:30-34; and Liu, et al., Proc. Natl. Acad. Sci.USA (1994) 91:6584-6588; Englebretsen, et al., Tet. Letts. (1995)36(48):8871-8874; Gaertner, et al., Bioconj. Chem. (1994) 5(4):333-338;Zhang, et al., Proc. Natl. Acad. Sci. USA (1998) 95(16):9184-9189; andTam, et al., WO 95/00846). Extended general chemical ligation andsynthesis also may be employed as disclosed in Kent, et al., WO98/28434.

Additionally, rapid methods of synthesizing assembled polypeptides viachemical ligation of three or more unprotected peptide segments using asolid support, where none of the reactive functionalities on the peptidesegments need to be temporarily masked by a protecting group, and withimproved yields and facilitated handling of intermediate products isdescribed in Canne, et al., WO/98/56807. Briefly, this method involvessolid phase sequential chemical ligation of peptide segments in anN-terminus to C-terminus direction, with the first solid phase-boundunprotected peptide segment bearing a C-terminal α-thioester that reactswith another unprotected peptide segment containing an N-terminalcysteine and a C-terminal thioacid. The techniques also permitssolid-phase native chemical ligation in the C- to N-terminus direction.Large polypeptides can also be synthesized by chemical ligation ofpeptide segments in aqueous solution on a solid support without need forprotecting groups on the peptide segments. A variety of peptidesynthesizers are commercially available for batchwise and continuousflow operations as well as for the synthesis of multiple peptides withinthe same run and are readily automated.

A ligation component also can be selected to contain moieties thatfacilitate and/or ease purification and/or detection. For example,purification handles or tags that bind to an affinity matrix can be usedfor this purpose. Many such moieties are known and can be introduced viapost-synthesis chemical modification and/or during synthesis. (See,e.g., Protein Purification Protocols, (1996), Doonan, S., ed., HumanaPress Inc.; Schriemer, et al., Anal. Chem. (1998) 70(8):1569-1575;Evangelista, et al., J. Chromatogr. B. Biomed. Sci. Appl. (1997)699(1-2):383-401; Kaufmann, M., J. Chromatogr. B. Biomed Sci. Appl.(1997) 699(1-2):347-369; Nilsson, et al, Protein Expr. Purif. (1997)11(1):1-16; Lanfermeijer, et al., Protein Expr. Purif. (1998) 12(1):29-37). For example, one or more unnatural amino acids having a chemicalmoiety that imparts a particular property that can be exploited forpurification can be incorporated during synthesis. Purificationsequences also can be incorporated by recombinant DNA techniques. Insome instances, it may be desirable to include a chemical or proteasecleavage site to remove the tag, depending on the tag and the intendedend use. An unnatural amino acid or chemically modified amino acid alsomay be employed to ease detection, such as incorporation of achromophore, hapten or biotinylated moiety detectable by fluorescencespectroscopy, immunoassays, and/or MALDI mass spectrometry.

Homogeneity and the structural identity of the desired membranepolypeptide and ligation label component synthesis products can beconfirmed by any number of means including immunoassays, fluorescencespectroscopy, gel electrophoresis, HPLC using either reverse phase orion exchange columns, amino acid analysis, mass spectrometry,crystallography, NMR and the like. Positions of amino acidmodifications, insertions and/or deletions, if present, can beidentified by sequencing with either chemical methods (Edman chemistry)or tandem mass spectrometry.

The lipid matrix component may include natural and/or synthetic lipidscapable of forming a lyotropic phase (e.g., liquid crystalline phaseupon interaction with water). Of particular interest are polar lipidssuch as phospholipids, lysophospholipids, sphingolipids, and glycolipidscapable of forming lamellar bilayers and other lipid aggregates, whichincludes mixtures of lipids capable of forming a lyotropic crystallinephase. Examples of such lipids include, but are not limited to,insoluble non-swelling amphiphiles, insoluble swelling amphiphiles, andvarious soluble amphiphiles capable of forming lyotropic liquidcrystalline phases. Examples of insoluble non-swelling amphiphilesinclude triglycerides, diglycerides, long chain protonated fatty acids,long chain normal alcohols, long chain normal amines, long chainaldehydes, phytols, retinols, vitamin A, vitamin K, vitamin E,cholesterol, desmosterol, sitosterol, vitamin D, unionized phosphatidicacid, sterol esters of very short chain acids, waxes in which eitheracid or alcohol moiety is less than 4 carbon atoms long (e.g., methyloleate), and ceremides. Examples of insoluble swelling amphiphilesinclude phosphatidylcholine, phosphatidylethanolamine,phosphatidylinositol, sphingomyelin, cardiolipid, plasmalogens, ionizedphosphatidic acid, cerebrosides, phosphatidylserine, monoglycerides,acid-soaps, α-hydroxy fatty acids, monoethers of glycerol, mixtures ofphospholipids and glycolipids extracted from cell membranes or cellularorganelles (plant glycolipids and sulfolipids), sulfocerebrosides,sphingosine (basic form). Examples of soluble amphiphiles capable offorming lyotropic liquid crystalline phases include sodium and potassiumsalts of long chain fatty acids, many of the ordinary anionic, cationic,and nonionic detergents, lysolecithin, palmitoyl and oleyl coenzyme A,and other long chain thioesters of coenzyme A, gangliosides, andsphingosine (acid form).

Preferred lipid matrices form stable membrane monolayers or bilayers andaggregate phases thereof. Of particular interest are lipids that formstable cubic phases, also referred to as a cubic lipidic phase of CLPmatrix (Lindblom, et al., supra). Examples of lipids capable of formingcubic phases include, but are not limited to, the following lipids:phosphatidylcholine (PC); dipalmitoylphosphatidylcholine (DPPC);1-palmitoyl-2-oleoylphosphatidylcholine (POPC);dioleoylphosphatidylcholine (DOPC); dilinoleoylphosphatidylcholine(DliPC); lysophosphatidylcholine (LPC); 1,palmityol-LPC (PaLPC);1-oleoyl-LPC (OILPC); 1,monoolein (MO); phosphatidylethanolamine (PE);plasmaenylethanolamine (PalE); glycerol acetal of plasmaenylethanolamine(GAPlaE); didodecylphosphatidylethanolamine (DDPE);dielaidoylphosphatidylethanolamine (DEPE);dioleoylphosphatidylethanolamine (DOPE);dilineoleoylphosphatidylethanolamine (DliPE);dioleoylphosphatidyl-N-monomethyl-ethylethanolamine (DOPE-Me);diphosphatidylglycerol (DPG); phosphatidylglycerol (PG);phosphatidylserine (PS); phosphatidylinositol (PI);monoglucolsyldiacylglycerol (MgluDG); monogalactosyldiacylglycerol(MgalDG); diglucosyldiacylglycerol (DgluDG);digalactosyl-diactylglycerol (DgalDG);dioleoylmonoglucosyldiacylglycerol (DOMGluDG);dioleoyldiglucosyldiacylglycerol (DODGluDG); glyceroldialkylnonitoltetraether (GDNT); and a glycerol-lipid mixture of 70% of GDNT withβ-D-glycopyranose linked to the nonitol group; 30% of glycerol dialkylglycerol tetraether with β-D-galactopyranosyl-β-D-galactopyranose linkedto one of the glycerol groups (GL).

Hydration, temperature and lipid composition can be varied to modulatethe liquid crystal structure of a lipid matrix. For example, cubic phaselipid systems that include mixtures of MO with1-palmitoyl-2-oleoyl-3-phosphatidylserine (PaOIPS) and DEPE/alamethicinform bicontinuous cubic phases under fully hydrated conditions withincertain concentration ratios and temperature ranges. In contrast, DOPEwith up to 10 mol % PaOIPS exists in the hexagonal phase at roomtemperature. Thus temperature and composition of the lipid membrane canbe used to control the crystalline phase. Such parameters are known formany lipid systems, or can be determined by various methods known in theart, including testing a serial array of mixtures with variousconcentrations of particular lipid components and water, and comparingthem over a range of temperatures. More specialized techniques also canbe employed to fine tune the cubic phases of a particular lipid/watersystem, such as determination of phase diagrams, X-ray diffraction, NMR,and polarized light microscopy and the like (Lindblom, et al., supra).

Lipids capable of forming cubic phases that have a certain water contentby weight percent (wt %) and minimum temperature (° C.) for formation ofthe cubic phase include, but are not limited to, the followinglipid/water systems: MO with 12-40 wt % water content and temperatureminimum of 20° C.; PaLPC with 40-46 wt % water content and temperatureminimum of 25° C.; OILPC with 20-25 wt % water content and temperatureminimum of 25° C.; Egg PC with 0-4 wt % water content and temperatureminimum of 75° C.; DOPC with 2-11 wt % water content and temperatureminimum of 60° C.; DliPC with 4 wt % water content and temperatureminimum of 55° C.; Egg PC+22-35 wt % sodium cholate with 22-26 wt %water content and temperature minimum of 22° C.; Egg PC+75-80 wt %diacylglycerol with excess water content and temperature minimum of 10°C.; Egg PC+85 wt % DliPE with excess water content and temperatureminimum of 40° C.; DOPE with 67 wt % water content and temperatureminimum of 25° C.; DOPE-Me with 67 wt % water content and temperatureminimum of 25° C.; DOPC+0-50 wt % DOPE with water content of 10 wt % andtemperature minimum of 70° C.; POPC+85-90 wt % DliPE with 29-41 wt %water content and temperature minimum of 7° C.; PE from P. regina withwater content of 35-50 wt % and temperature minimum of 40° C.; PE fromB. megaterium with 8-26 wt % water content and temperature minimum of58° C.; DDPE with water content of 10-16 wt % or greater than 33 wt %and temperature minimum of 75° C. or 115° C., respectively; DPG frombovine heart+29 wt % dibucane with 41 wt % water content and temperatureminimum of 7° C.; sodium sulfatide from human brain with 40-70% watercontent and temperature minimum of 20° C.; DOMGluDG from A. laidlawiiwith 7-15 wt % water content and temperature minimum of 0° C.;DOMGluDG+51 wt % DODGluDG from A. laidlawii with 10 wt % water contentand temperature minimum of 25° C.; MgalDG+31 wt % DgalDG from maizechloroplasts with 10-20 wt % water content and temperature minimum of60° C.; MgalDG 34-50 wt %+DgalDG from wheat chloroplasts with watercontent of 3-15 wt % and temperature minimum of 10° C.; GDNT with 7-13wt % water content and temperature minimum of 15° C.; GL with 0-11 wt %water content and temperature minimum of 60° C.; polar lipids from A.laidlawii with 18 wt % water content and temperature minimum of 65° C.;and polar lipids from S. solfactaricus with water content of 20 or 40 wt% and temperature minimum of 85° C. (Lindblom, et al., supra).

The lipids may be obtained from various sources including commercialsources or produced and prepared as micelles, liposome vesicles, CLPmatrices, or continuous lamellar membranes purified from cells followingstandard techniques known in the art. (See, e.g., Small, D. M., (1986)“The physical chemistry of lipids from alkenes to phospholipids,” In:Handbook of Lipid Research, vol. 4, pp. 1-672, D. Haccham, ed, PlenumPress New York; Winterhalter, et al., Chem. Phys. Lipids (1993)64(1-3):35-43; McNamee, M G., Biotechniques (1989) 7(5): 466-475;Albertsson et al., Methods Biochem Anal. (1982) 28: 115-150; Graham, JM., Methods Mol Biol. (1993) 19: 97-108; and Kinne-Saffran, et al.,Methods Enzymol. (1989) 172: 3-17; Larsson, K., J. Phys. Chem. (1989)93:7304-7314; Zumbuchl, et al., Biochimica et Biophysica Acta (198X)640:252-262; Erikson, et al., J. Phys. Chem. (1985) 91:846; Seddon, etal., Prog. Colloid Polym. Sci. (1990) 81:189; and U.S. Pat. No.5,554,650).

A lipid matrix for incorporation of a membrane polypeptide is selectedfor compatibility with the polypeptide. In particular, a membranepolypeptide known to be stable over a particular temperature range isselected for incorporation in a lipid matrix having a compatibletemperature profile. By way of example, when a CLP matrix is employed,the temperature profile for formation of the cubic phase is selected soas to be compatible with incorporation and stability of the membranepolypeptide. A CLP comprising a MO/water system is suitable for mostmembrane polypeptides stable at room temperature (˜25° C.). Thermostablemembrane polypeptides on the other hand may be more suited for insertionin CLP comprising a DOPE/water system having a temperature minimumprofile for formation of a cubic phase of about 70° C.

Incorporation of a membrane polypeptide ligation component into a lipidmatrix of interest can be accomplished in vitro and/or in vivo. Whenexpressed ribosomally in a cell or cell free system containing a lipidmembrane, the membranes and polypeptides associated therewith can beisolated together, further refined if desired, or the polypeptidesseparated from the membranes for subsequent reconstitution in apreformed lipid matrix following standard techniques in the art. (See,e.g., Hubbel, et al., “Membrane Protein Structure: ExperimentalApproaches,” S. White, ed., Oxford Univ. Press, London, 1994; Kahn, etal., Biochemistry (1992) 31:6144-6151; Nowak, et al., Science (1995)268:439-442; Turcatti, et al., J. Biol. Chem. (1996)271(33):19991-19998; Okumura, et al., Biochimica et Biophysica Acta(1994) 1194(2):335-340; Mimms, Biochemistry (1981) 20:833-839; and U.S.Pat. No. 4,515,736). For directing a specific membrane polypeptide to aparticular lipid matrix, a native or artificial targeting sequence thatfacilitates localization of the target polypeptide to a particular cellmembrane of interest also may be included. (See., e.g., Pelham, et al.,Cell (1993) 75:603-605; and Magee, et al., (1994) Protein Targeting: APractical Approach, D. Rickwood and B. D. Hames, eds., Oxford UniversityPress, Oxford).

When incorporated in a preformed lipid matrix, a target membranepolypeptide can be reconstituted following standard techniques in theart, including a preformed lipid matrix of native cell membranes,liposome, micelles, or CLPs. (See, e.g., Cladera, et al., Eur. Biochem.(1997) 243(3):798-804; Takahashi, et al., Nature Struct. Biol. (1997)4:44-50; Das, T K, J. Phys. Chem. (1996) 100:20143-20147; Angrand, etal., Eur. J. Biochem. (1997) 250(1):168-176; Zardeneta et al., Anal.Biochem. (1994) 223(1):1-6; Landau, et al., J. Amer. Chem. Soc. (1993)115:2102-2106; Portman, et al., J. Phy. Chem. (1991) 95:8437-8440; andMariani, et al., J. Mol. Biol. (1988) 204:165-189). In particular, atarget membrane polypeptide can be reconstituted in a preformed lipidmatrix by controlled solubilization and/or lipid extraction techniques.For instance, the membrane polypeptide can be added to an appropriatesolvent/detergent system, and extracts admixed with a preformed lipidmatrix that contains or is devoid of solvent/detergent in an amount thatpermits solubilization and insertion of the membrane polypeptide in thelipid matrix. One or more reconstitution conditions may be adjusted tooptimize the process, such as temperature, pH, water and/or organicsolvent content, ion concentration, reducing or oxidizing reagents ifpresent, as well as ratios of detergent to lipid, detergent topolypeptide, and lipid to polypeptide. An example is admixing a lipidmatrix and membrane polypeptide in an appropriate buffer system withserial amounts of a detergent, and monitoring membrane polypeptideincorporation at each step of the reconstitution process to assessoptimal buffer/detergent profiles for a particular lipid-membranepolypeptide reconstitution system. Examples of lipid solubilizationreagents suitable for reconstitution analyses include SDS (sodiumdodecyl sulfate), Triton-X100, Ammonyx-LO (N,N-dimethyl lauryl amineoxide), sodium cholate, taurocholate, sucrose monolaurate,dodecylmaltoside, CHAPS(3-(3-cholamidopropyl)-dimethylammonio-1-propanesulfonate), CHAPSO(3-[(3-cholamidopropyl) dimethylammonio]-2-hydroxy-1-propane sulfonate),octylglucoside, octylglucopyranoside and the like. (See, e.g., Rigaud,et al., Biochimica et Biophysica Acta (1995) 1231(3):223-246; Yang Q, etal., Anal. Biochem. (1994) 218(1):210-221; Scotto, et al., Biochemistry(1987) 26(3):833-839). If necessary, detergent can be removed followingreconstitution and the content and/or activity of the reconstitutedsystem characterized by various methods known in the art.

Reconstitution by lipid extraction can be performed through addition ofisolated membrane polypeptide to a lipid matrix in an appropriateorganic solvent system, such as a hexane and buffered aqueous solution,for separation of aqueous soluble phase from lipid-organic solventsoluble phase. As with the solvent/detergent solubilization methodabove, one or more reconstitution conditions may be adjusted to optimizethe process, such as temperature, pH, water and/or organic solventcontent, ion concentration, reducing or oxidizing reagents if present,as well as ratios of detergent to lipid, detergent to polypeptide, andlipid to polypeptide. The extracted lipid phase containing the membranepolypeptide can then be employed to form lipid monolayers, bilayers,liposome, micelles or CLPs by standard techniques such as sonication,layering, extrusion, centrifugation and the like depending on the lipidmatrix to be obtained. Any suitable technique for reconstitution ofmembrane polypeptides in a lipid matrix by lipid extraction can be used.(See., e.g., Montal, et al., Q. Rev. Biophys. (1981) 14:1-79; Ayala, etal., Biochimica et Biophysica Acta (1985) 810(2):115-122; Montal, etal., Proc. Natl Acad Sci. USA (1990) 87:6929; Puu, et al., BiosenBioelectron (1995) 10(5):463-476). For instance, the solvent system canbe selected for a particular lipid-membrane polypeptide to optimizeextraction, such as described above for obtention and preparation oflipids. As with the solubilization method of incorporating a membranepolypeptide in a lipid matrix, serial amounts of a chosen solvent withor without detergent and/or salts can be employed to monitor and selectan optimal lipid extraction system to obtain an extracted lipid phasecontaining the membrane polypeptide.

The ligation label can be optionally added to the lipid matrix before,during and/or after incorporation of the membrane polypeptide dependingon the ligation component pairing and its intended end use. When anaqueous-soluble ligation label is used, the label may be added to alipid matrix that is already incorporated with a target membranepolypeptide. A soluble ligation label also can be added before and/or inconjunction with incorporation of the membrane polypeptide component inthe lipid matrix, for instance, inside an aqueous compartment of aliposome, micelle or CLP. The latter is particularly advantageous whenthe membrane polypeptide is capable of spontaneous insertion in apreformed lipid matrix comprising a ligation label. Similarly, alipid-soluble ligation label may be added before, during and/or afterthe membrane polypeptide is incorporated. As an example, when theligation label comprises a lipid-embedding anchor domain capable offorming a complex with a lipid-embedding anchoring domain of a membranepolypeptide component, it may be desirable to incorporate the ligationlabel with the membrane polypeptide component before ligation so as toobtain a complex or bundle of self-assembled, yet unconnected domains.Incorporation of the membrane polypeptide and ligation components beforeligation can be used to assist in insertion and/or folding of individualpolypeptide segments or domains upon their interaction in the lipidmatrix as well as promote proper positioning of a target ligation site.This approach also is particularly useful when the target ligation siteis a compatible ligation component pairing designed to generate amodular multi-pass transmembrane polypeptide upon ligation of anextracellular and/or an intracellular loop that joins the inserteddomains. Another example is the expression and insertion of the ligationlabel in a lipid membrane of a cell or cell free system, followed bysubsequent reconstitution of the cell membrane containing the ligationlabel with a membrane polypeptide. To maintain the desired lipid matrixsystem following incorporation of the membrane polypeptide and/orligation label, conditions such as temperature, pH, water and/or organicsolvent content, ion concentration, reducing or oxidizing reagents ifpresent, as well as ratios of detergent to lipid, detergent topolypeptide, and lipid to polypeptide can be adjusted as needed.Aliquots can be removed at specific time intervals to monitor theincorporation and integrity of the following standard techniques orthose described herein.

When the membrane polypeptide and ligation label are reconstitutedtogether, one or more of the ligation components can include a linker orcapping sequence having one or more cleavage sites positioned forgenerating a chemically reactive ligation site moiety upon cleavage, andthus used to control the presence or absence of reactive groups neededfor the ligation reaction. Additionally, reagents or buffer conditionscan be utilized to control the presence and reactivity of such reactivegroups. By way of example, buffer conditions with a pH of less than 6.5also can be utilized to impede native chemical ligation. As anotherexample, when the ligation components are designed for native chemicalligation, and thus collectively provide a complementary ligationcomponent pairing comprising an unprotected N-terminal cysteine and aC-terminal αCOSR group, such as created after cleavage and generation ofa cysteine ligation site, a thiol reducing agent such asβ-mercaptoethanol may be optionally included if one chooses to delay orimpede a ligation reaction. Of course the presence or absence ofdisulfide bonds and folding of the membrane polypeptide in the lipidmatrix are taken into consideration before adding a thiol reducingagent. In general, if a disulfide bond is necessary for maintaining aproperly inserted and folded membrane polypeptide, and the disulfide issensitive to the thiol reducing agent, then the ligation label can beadded just prior to ligation, thereby minimizing the need to add areducing reagent to delay or impede ligation.

However the ligation label component is provided to the lipid matrixsystem, one of ordinary skill in the art will recognize that the methodand order of incorporation can be adjusted for a given end use, providedthat the integrity and compatibility of the lipid matrix-embeddedmembrane polypeptide and ligation label component pairings aremaintained and capable of subsequent ligation as exemplified in any oneor more of Schemes 1-2.

Once a membrane polypeptide and/or ligation label component isincorporated in a lipid matrix of interest, the matrix can be stored orfurther processed for ligation. For ligation, the lipidmatrix-incorporated membrane polypeptide may be provided in a solutionphase or attached to a solid support phase matrix. This includes liquidsolutions, gels, slurries, affinity matrix support systems and the like.If the membrane polypeptide or ligation label includes a cleavage siteadjacent to a residue targeted for chemoselective chemical ligation,cleavage is performed prior to the ligation reaction. Chemical orprotease cleavage is performed by admixing a cleavage reagent specificfor the cleavage site and incubating the mixture under the appropriateconditions and time so as obtain cleavage. Many site-specific cleavagereagents are well known and can be produced or obtained from commercialvendors. The cleaving reagent and reaction by-products can then beneutralized or removed prior to ligation if desired, so as to avoid anyinterference with the ligation reaction. Extent of cleavage afterincubation and generation of lipid matrix-incorporated cleavage productcan be determined by any number of techniques known in the art ordescribed herein. Of course it will be understood that the product ofthe cleavage reaction targeted for chemical ligation will contain anunprotected amino acid residue amenable to a particular type of ligationchemistry, whether the product is the ligation label and/or the membranepolypeptide. By way of example, a cleavage product designed for nativechemical ligation will preferably contain an unprotected N-terminalcysteine, or in some instances only an appropriately positionedunprotected amino acid is needed for native chemical ligation to amembrane polypeptide or ligation label donating a C-terminal thioestermoiety.

For the ligation reaction, reaction conditions are selected thatmaintain the desired interaction of the lipid matrix-embedded membranepolypeptide and ligation label components. Routine adjustments-toidentify optimal conditions can be made, for instance, by varyingindividual reagents, concentrations, temperatures and the like. Forexample, reaction conditions may be adjusted to optimize ligation, suchas temperature, pH, water and/or organic solvent content, ionconcentration, reducing or oxidizing reagents if present, as well asratios of detergent to lipid, detergent to polypeptide, and lipid topolypeptide. Addition or exclusion of reagents that solubilize the lipidmatrix and/or the membrane polypeptide to different extents may furtherbe used to control the specificity and rate of the desired ligationreaction. One of ordinary skill will recognize that these conditions areselected based on a given ligation chemistry utilized, and thusconditions appropriate to that chemistry. Such conditions and ranges ofconditions are readily determined by one of ordinary skill in the art.

Any number of techniques can monitor formation and the homogeneity ofthe desired lipid matrix-membrane polypeptide and/or ligation labelcomponent system, as well as formation of a desired ligation product.These techniques include assays that detect activity of the insertedmembrane polypeptide, fluorescence spectroscopy, mass spectrometry,affinity matrix and ligand binding assays, NMR, circular dichroism (CD),scanning densitometry, calorimetry, and various chromatographytechniques such as electrophoresis, affinity chromatography, HPLC usingeither reverse phase or ion exchange columns, and the like.

A lipid matrix comprising a pre-ligation or post-ligation product can bestored for later use or directly employed in an assay. When stored, themethod of storage is selected to retain stability of the lipid matrixand its incorporated components. This includes storage in a frozen form,in the form of a lyophilized powder, crystal, gel, slurry, liquid, or inany other suitable form, including storage on a support matrix such as afilm or affinity matrix. For example, a lipid matrix fraction derivedfrom a native cell membrane, unless highly purified, is typically frozenif intended for long term storage. Artificial lipid matrices such asliposomes and CLPs are somewhat more amenable to a range of storageoptions, particularly CLPs, which may be stored as viscous gels underconditions that favor the cubic phase of the lipid system. When providedas a closed vesicle, such as a liposome, aggregation and fusion may bereduced by adding negative and/or positively charged components to thecomposition following standard techniques. Introducing lipids that havea higher transition temperature, such as cholesterols or saturated fattyacid containing phospholipids, also may reduce leakage of closedvesicles. Additives such as trehalose may also be employed as acryoprotector. Of course any suitable method for storing preformed lipidsystems known in the art can be used and storage stability in generalcan be improved by regulating temperature and moisture content, reducingcontacts with raw materials or with oxidants, such as by storing underan inert gas, dispersing the lipid matrices in a neutral buffer systemand/or eliminating residual solvents.

For employment in an assay, the chemical ligation methods andcompositions of the invention can be utilized in a screening assay ofthe invention. Screening assays of the invention are characterized bybinding of a ligand to a target lipid matrix-embedded membranepolypeptide produced by one or more of the methods and compositions ofthe invention.

In a preferred embodiment, at least one ligation component is providedwith one or more detectable moieties, also referred to as a detectablelabel or probe, such as an isotope, hapten, or chromophore includingfluorophores, heavy metal complexes with aromatic ligands or chelateligands and the like. More preferably, at least one first detectablelabel is incorporated into the membrane polypeptide by a chemoselectivechemical ligation approach exemplified in any one or more of Schemes1-2. If desired, one or more of a second detectable label can beincorporated into the membrane polypeptide, the lipid matrix, and/or aligand for the polypeptide by any number of techniques in addition toSchemes 1-2. As described above, a detectable moiety can be incorporatedin a ligation component during and/or after synthesis, including afterligation in the lipid matrix, by any technique suitable for suchpurpose, provided the reactivity, selectivity and stability of the lipidmatrix-incorporated membrane polypeptide and/or ligation componentsystem exemplified in any one or more of Schemes 1-2 is substantiallymaintained. As also described above, one or more residues targeted forcovalent attachment of a detectable label can be selected during thedesign and screening of the compatible ligation component pairings.

Chemical synthesis is the preferred method to incorporate a detectablelabel. In this embodiment, chemical synthesis is utilized to incorporateat least one detectable label in a pre-ligation component exemplified inany one or more of Schemes 1-2. In this way the resulting lipid-membraneembedded ligation product can be designed to contain one or moredetectable labels at pre-specified positions of choice. Isotopic labelsdetectable by NMR are of particular interest. Also of particularinterest is the incorporation of one or more unnatural amino acidscomprising a detectable label at one or more specific sites in a targetligation component of interest. By unnatural amino acid is intended anyof the non-genetically encoded L-amino acids and D-amino acids that aremodified to contain a detectable label, such as photoactive groups, aswell as chromophores including fluorophores and other dyes, or a haptensuch as biotin. Unnatural amino acids comprising a chromophore andchemical synthesis techniques used to incorporate them into a peptide orpolypeptide sequence are well known, and can be used for this purpose.For example, it may be convenient to conjugate a fluorophore to theN-terminus of a resin-bound peptide before removal of other protectinggroups and release of the labeled peptide from the resin. Fluorescein,eosin, Oregon Green, Rhodamine Green, Rhodol Green,tetramethylrhodamine, Rhodamine Red, Texas Red, coumarin and NBDfluorophores, the dabcyl chromophore and biotin are all reasonablystable to hydrogen fluoride (HF), as well as to most other acids, andthus suitable for incorporation via solid phase synthesis. (Peled, etal., Biochemistry (1994) 33:7211; Ben-Efraim, et al., Biochemistry(1994)33:6966). Other than the coumarins, these fluorophores also arestable to reagents used for deprotection of peptides synthesized usingFmoc chemistry (Strahilevitz, et al., Biochemistry (1994)33:10951). Thet-Boc and α-Fmoc derivatives of ε-dabcyl-L-lysine also can be used toincorporate the dabcyl chromophore at selected sites in a polypeptidesequence. The dabcyl chromophore has broad visible absorption and canused as a quenching group. The dabcyl group also can be incorporated atthe N-terminus by using dabcyl succinimidyl ester (Maggiora, et al., J.Med Chem (1992) 35:3727). EDANS is a common fluorophore for pairing withthe dabcyl quencher in FRET experiments. This fluorophore isconveniently introduced during automated synthesis of peptides by using5-((2-(t-Boc)-γ-glutamylaminoethyl) amino) naphthalene-1-sulfonic acid(Maggiora, et al., J. Med. Chem. (1992) 35:3727). Anα-(t-Boc)-γ-dansyl-L-lysine can be used for incorporation of the dansylfluorophore into polypeptides during chemical synthesis (Gauthier, etal., Arch Biochem. Biophys. (1993) 306:304). As with EDANS fluorescenceof this fluorophore overlaps the absorption of dabcyl. Site-specificbiotinylation of peptides can be achieved using the t-Boc-protectedderivative of biocytin (Geahlen, et al., Anal. Biochem. (1992) 202:68),or other well known biotinylation derivatives such as NHS-biotin and thelike. Racemic benzophenone phenylalanine analog also can be incorporatedinto peptides following its t-Boc or Fmoc protection (Jiang, et al.,Intl. J. Peptide Prot. Res. (1995) 45:106). Resolution of thediastereomers can be accomplished during HPLC purification of theproducts; the unprotected benzophenone also can be resolved by standardtechniques in the art. Keto-bearing amino acids for oxime coupling,aza/hydroxy tryptophan, biotyl-lysine and D-amino acids are among otherexamples of unnatural amino acids that can be utilized. It will berecognized that other protected amino acids for automated peptidesynthesis can be prepared by custom synthesis following standardtechniques in the art.

Other detectable labels can be incorporated into the membranepolypeptide, the lipid matrix, and/or a ligand for the polypeptide by atechnique in addition to those exemplified in Schemes 1-2. In thisembodiment, the detectable moiety typically is introduced post-chemicalligation. This can be done by chemical modification using a reactivesubstance that forms a covalent linkage once having bound to a reactivegroup of the target molecule. For example, a peptide or polypeptideligation component can include several reactive groups, or groupsmodified for reactivity, such as thiol, aldehyde, amino groups, suitablefor coupling the detectable label by chemical modification (Lundblad, etal., In: Chemical Reagents for Protein Modification, CRC Press, BocaRaton, Fla., (1984)). Site-directed mutagenesis and/or chemicalsynthesis also can be used to introduce and/or delete such groups from adesired position. Any number of detectable labels includingbiotinylation probes of a biotin-avidin or streptavidin system,antibodies, antibody fragments, carbohydrate binding domains,chromophores including fluorophores and other dyes, lectin, nucleic acidhybridization probes, drugs, toxins and the like, can be coupled in thismanner. For instance, a low molecular weight hapten, such a fluorophore,digoxigenin, dinitrophenyl (DNP) or biotin, can be chemically attachedto the membrane polypeptide or ligation label component by employinghaptenylation and biotinylation reagents. The haptenylated polypeptidethen can be directly detected using fluorescence spectroscopy, massspectrometry and the like, or indirectly using a labeled reagent thatselectively binds to the hapten as a secondary detection reagent.Commonly used secondary detection reagents include antibodies, antibodyfragments, avidins and streptavidins labeled with a fluorescent dye orother detectable marker.

Depending on the reactive group, chemical modification can be reversibleor irreversible. A common reactive group targeted in peptides andpolypeptides are thiol groups, which can be chemically modified byhaloacetyl and maleimide labeling reagents that lead to irreversiblemodifications and thus produce more stable products. For instance,reactions of sulfhydryl groups with α-haloketones, amides, and acids inthe physiological pH range (pH 6.5-8.0) are well known and allow for thespecific modification of cysteines in peptides and polypeptides(Hermason, et al., In: Bioconjugate Techniques, Academic Press, SanDiego, Calif., pp. 98-100, (1996)). Covalent linkage of a detectablelabel also can be triggered by a change in conditions, for example, inphotoaffinity labeling as a result of illumination by light of anappropriate wavelength. For photoaffinity labeling, the label, which isoften fluorescent or radioactive, contains a group that becomeschemically reactive when illuminated (usually with ultraviolet light)and forms a covalent linkage with an appropriate group on the moleculeto be labeled. An important class of photoreactive groups suitable forthis purpose is the aryl azides, which form short-lived but highlyreactive nitrenes when illuminated. Flash photolysis of photoactivatableor “caged” amino acids also can be used for labeling peptides that arebiologically inactive until they are photolyzed with UV light. Differentcaging reagents can be used to modify the amino acids, such derivativesof o-nitrobenzylic compounds, and detected following standard techniquesin the art. (Kao, et al., “Optical Microscopy: Emerging Methods andApplications,” B. Herman, J. J. Lemasters, eds., pp. 27-85 (1993)). Thenitrobenzyl group can be synthetically incorporated into thebiologically active molecule via an ether, thioether, ester (includingphosphate ester), amine or similar linkage to a hetero atom (usually O,S or N). Caged fluorophores can be used for photoactivation offluorescence (PAF) experiments, which are analogous to fluorescencerecovery after photobleaching (FRAP). Those caged on the ε-amino groupof lysine, the phenol of tyrosine, the γ-carboxylic acid of glutamicacid or the thiol of cysteine can be used for the specific incorporationof caged amino acids in the sequence. Alanine, glycine, leucine,isoleucine, methionine, phenylalanine, tryptophan and valine that arecaged on the α-amine also can be used to prepare peptides that are cagedon the N-terminus or caged intermediates that can be selectivelyphotolyzed to yield the active amino acid either in a polymer or insolution. (Patchornik, et al., J. Am Chem Soc (1970) 92:6333). Spinlabeling techniques of introducing a grouping with an unpaired electronto act as an electron spin resonance (ESR) reporter species may also beused, such as a nitroxide compound (—N—O) in which the nitrogen formspart of a sterically hindered ring (Oh, et al., supra).

Selection of a detectable label system generally depends on the assayand its intended use. In particular, the chemical ligation methods andcompositions of the invention can be employed in a screening assay ofthe invention characterized by binding of a ligand to a lipidmatrix-embedded membrane polypeptide comprising a ligation label joinedthrough a covalent bond to the membrane polypeptide ligation site. Theseinclude diagnostic assays, screening new compounds for drug development,and other structural and functional assays that employ binding of aligand to a prefolded membrane polypeptide. The ligands may be derivedfrom naturally occurring ligands or derived from synthetic sources, suchas combinatorial libraries. Screening methods of particular interestinvolve detection of ligand binding to a lipid matrix-embedded membranepolypeptide, as produced by a method of the invention, by fluorescencespectroscopy.

In a preferred embodiment, screening for binding of a ligand to a lipidmatrix-embedded membrane polypeptide comprising one or more chromophoresis performed in an assay characterized by detecting fluorescenceresonance energy transfer (FRET). Ligand binding to the lipidmatrix-embedded membrane polypeptide can be measured by any number ofmethods known in the art for FRET analyses, including steady state andtime-resolved fluorescence by monitoring the change in fluorescenceintensity, emission energy and/or anisotropy, for example, throughenergy transfer from a donor moiety to an acceptor moiety of the FRETsystem. (See, e.g., Wu, et al., Analytical Biochem. (1994) 218:1-13).FRET assays allow not only distance measurements, but also resolution ofthe range of donor-to-acceptor distances. FRET also can be used to showthat the membrane polypeptide exists alternately in a singleconformational state, or with a range of donor-to-acceptor distanceswhen in a different state, such as when bound to a ligand.

For FRET assays, the lipid-matrix-incorporated membrane polypeptide isdesigned to contain at least one chromophore of a donor-acceptor system.The donor molecule is always a fluorescent (or luminescent) one fordetection. The acceptor molecule can be either fluorescent ornon-fluorescent. Thus for a donor-acceptor system, at least twochromophores are provided: the first is provided by the lipidmatrix-embedded membrane polypeptide; the second can be provided by themembrane polypeptide, the lipid matrix, or by a ligand for thepolypeptide.

More than one donor-acceptor pairing may also be included. For example,the membrane polypeptide may contain one or more donor and/or acceptormolecules, and preferably at least one donor molecule comprising achromophore. A ligand may comprise one or more donor or acceptormolecules as well. The lipid membrane matrix also may contain one ormore donor or acceptor molecules, as well as secondary moleculesincorporated into the lipid and/or solvent accessible channels formed bythe matrix that are ligands of the target polypeptide of interest, suchas G-proteins when the target membrane polypeptide comprises part or allof a seven-transmembrane receptor system.

In a preferred embodiment, the membrane polypeptide comprises one ormore donor molecules and a ligand for the membrane polypeptide comprisesone or more acceptor molecules. The ligands can be small organicmolecules, peptides, peptide mimetics, constrained peptides,polypeptides and the like. They may be derived from naturally occurringligands or derived from synthetic sources, such as combinatoriallibraries. When a ligand is a peptide or polypeptide, it can be preparedwith a chromophore label using various chemistries, such as thosedescribed herein. Of particular interest are modular “cross-over”polypeptide ligands, which can be constructed to comprise one or morechromophores produced by combining one or more functional modules from afirst polypeptide and at least one second polypeptide. Synthesis ofmodular polypeptides is described in Kent, et al., WO 99/11655.

Ligands of particular interest compete for binding to a ligand bindingsite on a lipid matrix-embedded membrane polypeptide produced by any oneor more of the methods and/or compositions exemplified in Schemes 1-2.These ligands can be employed in an additional method of the inventionthat involves contacting a lipid matrix-embedded membrane polypeptidewith two or more different ligands that compete for binding to a ligandbinding site of the membrane polypeptide. One or more of the differentcompetition ligands can be labeled for detection purposes. As anexample, the membrane polypeptide can be labeled with a firstchromophore and a competition ligand can be labeled with a secondchromophore of a donor-acceptor system. Alternatively, the membranepolypeptide or the lipid matrix can provide the second chromophore.Competition for ligand binding to the polypeptide is monitored by FRETanalysis, including monitoring changes in intrinsic fluorescence of themembrane polypeptide upon binding ligand, and/or changes in theproperties of a fluorescent-labeled ligand upon binding, and thus ligandbinding can be detected and characterized.

When choosing a chromophore donor-acceptor pair for FRET, positioning ofthe first chromophore in a target polypeptide is designed to be within asufficient distance of a second chromophore to create a donor-acceptorfluorescence resonance energy transfer system. For instance, energytransferred from the donor to an acceptor involves coupling of dipolesin which the energy is transferred over a characteristic distance calledthe Forster radius (R_(o)), which is defined as the distance at whichenergy transfer efficiency is 50% (i.e., distance at which 50% ofexcited donors are deactivated by FRET). This distance is referred toherein as the Forster distance. These distances range from about 10 to100 Angstroms (Å), which is comparable to the diameter of many proteinsand comparable to the thickness of membranes. Intrinsic tryptophan ortyrosine sometimes may be used as chromophores in distance measurements,but in most cases the Forster distance is limited to above 30 Å.However, an acceptor molecule comprising clusters of acceptors with highmolar absorption coefficient for each acceptor may achieve a furtherextension of Forster distance. Thus average distances over 100Å can bemeasured. As the Forster distances can be reliably calculated from theabsorption spectrum of the acceptor and the emission spectrum of thedonor, FRET allows determination of molecular distances. Once theForster distance is known, the extent of energy transfer can be used tocalculate the donor-to-acceptor distance.

Donor-acceptor chromophores applicable for biological molecules, and forwhich Forster distances are known when paired, include but are notlimited to the following chromophores: ANAI (2-anthracenceN-acetylimidazole); BPE (B-phycoerythrin); CF (caboxyfluoresceinsuccinimidyl ester); CPM(7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin); CY5(carboxymethylindocyanine-N-hydroxysuccinimidyl ester, diI-C₁₃,1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine; diO-C₁₄,3,3′-ditetradecyloxacarbocyanine); DABM(4-dimethylaminophenylazo-phenyl-4′-maleimide); DACM((7-(dimethylamino)coumarin-4-yl)-acetyl); DANZ (dansylaziridine); DDPM(N-(4-dimethylamino-3,5-dinitrophenyl)maleimide); DMAMS(dimethylamino-4-maleimidostilbene); DMSM(N-(2,5-dimethoxystiben-4-yl)-maleimide); DNP (2,4-dinitrophneyl); ε-A(1,N⁶-ethenoadenosine); EIA (5-(iodoacetetamido) eosin); EITC (eosinthiosemicarbazide); F₂DNB (1,5-difluro-2,4′-dinitrobenzene); F₂DPS(4,4′-difluoro-3,3′-dinitrophenylsulfone); FITC(fluorescein-5-isothiocyanate); FM (fluorescein-5-maleimide); FMA(fluorescein mercuric acetate); FNAI (fluorescein N-acetylimidazole);FTS (fluorescein thiosemicarbazide); IAANS(2-(4′-iodoacetamido)amino)naphthalene-6-sulfonic acid); IAEDANS(5-(2-((iodoacetyl)amino)ethyl)amino)-naphthlene-1-sulfoni acid); IAF(5-iodoacetamidofluorescein); IANBD(N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole);IPM (3(4-isothiocyanatophenyl)7-diethyl-4-amino-4-methylcoumarin); ISA(4-(iodoacetamido)salicylic acid); LRH (lissaminerhodamine); LY (Luciferyellow); mBBR (monobromobimane); MNA ((2-methoxy-1-naphthyl)-methyl);NAA (2-naphthoxyacetic acid); NBD (7-nitro-2,1,3-benzoxadiazol-4-yl);NCP (N-cyclohexyl-N′-(1-pyrenyl)carbodiimide); ODR (octadecylrhodamine);PM (N-(1-pyrene)-maleimide); SRH (sulforhodamine); TMR(tetramethylrhodamine); TNP (trinitrophenyl); TR (Texas red); BODIPY((N1-B)-N1′-(difluoroboryl)-3,5′-dimethyl-2-2′-pyrromethene-5-propionicacid, N-succinimidyl ester); and lanthanide-ion-chelates such as aniodoacetamide derivative of the Eu³⁺-chelate of N-(p-benzoicacid)diethylenetriamine-N,N′,N′-tetraacetic acid (DTTA); Eu-DTPA-cs 124and Tb-DTPA-cs 124 (Eu-chelate and Tb-chelate ofdiethylenetriaminepentaacetic acid, carbostyril 124); Eu-TTHA-cs124(Eu-chelate of triethylenetetraaminehexaacetic acid, carbostyril 124);Eu-DOTA (Eu-chelate of 1,4,7,10 tetraazacyclodedecane N,N′,N″,N′″tetraacetic acid); Eu-DTPA-AMCA (Eu-chelate ofdiethylenetriaminepentaacetic acid, 7-amino 4-methyl coumarin); and Cy5and Cy5.5 (cyanine dye cy5 and cy5.5).

Since energy transfer measurement is most sensitive to distancevariation when donor-acceptor separation is close to their Forsterdistance, the membrane polypeptide comprising the first chromophore of adonor-acceptor pair system is selected or engineered so that the firstand second chromophores approach or are at the Forster distance. Table 1shows some typical Forster distances of donor-acceptor pairs, withselect ones shown as measured in H₂O and D₂O (Selvin et al., Proc. Natl.Acad. Sci. USA (1994) 91:10024-10028; Li, et al., J. Amer. Chem. Soc.(1995) 117:8132-8138; and Heyduk, et al., Anal. Biochem. (1997)248:216-227) for comparison.

TABLE 1 DONOR ACCEPTOR FORSTER DISTANCE (Å) FluoresceinTetramethyllrhodamine 55 IAEDANS Fluorescein 46 EDANS DABCYL 33Fluorescein Fluorescein 44 BODIPY FL BODIPY FL 57 Tb-DTPA-cs124 TAMRA 60(H₂O)/65(D₂O) Eu-TTHA-cs124 Cy5 68.6(H₂O)/73.5(D₂O) Eu-DTPA-cs124 Cy556(H₂O)/70(D₂O) Eu-DOTA Cy5.5 62(H₂O)/76(D₂O) Eu-DTPA-AMCA Cy555(H₂O)/61.4(D₂O)

Extensive compilations of Forster distances for various donor-acceptorpairs and their specific applications in FRET analysis of biologicalmolecules including peptides, polypeptides, carbohydrates and lipids arewell known in the art. (See, e.g., Wu, et al., supra; Berlman, et al.,(1973) Energy Transfer Parameters of Aromatic Compounds, Academic Press,New York; Van der Meer, et al., (1994) “Resonance Energy Transfer Theoryand Data,” VCH Publishers; Fairclough, et al., J. Muscle Res. CellMotility (1987) 8:97; des Remedios, et al., Meth. Enzymol. (1978)48:347). These Forster distances are used as a general guide whenselecting a particular donor-acceptor pair.

In addition to selecting donor and acceptor moieties that are in closeproximity (typically 10-100 Å) and approach or are at the Forsterdistance, the FRET chromophore pairs are selected so that the absorptionspectrum of the acceptor overlaps the fluorescence emission spectrum ofthe donor, and the donor and acceptor transition dipole orientations areapproximately parallel. Moreover, for anisotropy assays in which twoidentical chromophores are employed, where one is provided by themembrane polypeptide and a second by a ligand, the chromophores arepreferably positioned so that tumbling of the donor or acceptor moietyis minimized. In a preferred embodiment, at least one chromophore ispositioned in a rigid portion of the lipid matrix-embedded membranepolypeptide and/or ligation label component, such as in an α-helix orβ-sheet portion. A chromophore positioned and anchored between twocysteine residues about 2-3 Å apart in an α-helix or β-sheet isparticularly suited for this purpose. An advantage of reducingchromophore tumbling is increased sensitivity in FRET detection byreducing background noise in the spectrum.

For most applications, the donor and acceptor dyes are different, inwhich case FRET can be detected by the appearance of sensitizedfluorescence of the acceptor (acceptor enhancement), by quenching ofdonor fluorescence (donor quenching), or fluorescence polarization(anisotropy). When the donor and acceptor are the same, FRET istypically detected by anisotropy. For instance, donor quenching(quenching of fluorescence) can be used to detect energy transfer.Excitation is set at the wavelength of donor absorption and the emissionof donor is monitored. The emission wavelength of donor is selected suchthat no contribution from acceptor fluorescence is observed. Thepresence of acceptor quenches donor fluorescence. A wide variety ofsmall molecules or ions act as quenchers of fluorescence, that is, theydecrease the intensity of the emission. These substances include iodide,oxygen, chlorinated hydrocarbons, amines, and disulfide groups. Theaccessibility of fluorophores to quenchers is widely used to determinethe location of probes on macromolecules, or the porosity of proteinsand membranes to the quenchers. For example, the intensity ofpolypeptide- and membrane-bound fluorophores in the presence ofdissolved water-soluble quenchers can be measured by donor quenching.Lipid-soluble quenchers, such as brominated fatty acids, also can beadded to assess the interior acyl side chain region of membranes.

Acceptor enhancement detection techniques can be used when an acceptoris fluorescent, and its fluorescence intensity is enhanced when energytransfer occurs (with excitation into the donor). This providesadditional methods to visualize energy from a fluorescence spectrum. Inan emission spectrum, one excites at the wavelength of donor absorptionand observes the intensity increase of acceptor. In an excitationspectrum, one sets detection at the acceptor emission wavelength andobserves enhancements of intensity at a wavelength range where donorabsorbs.

Anisotropy (or fluorescence polarization) analysis using FRET is ofparticular interest, for instance, where two identical chromophores areattached to a lipid matrix-embedded membrane polypeptide and awater-soluble ligand. The polarization properties of light and thedependence of light absorption on the alignment of the fluorophores withthe electric vector of the incident light provide the physical basis foranisotropic measurements. Fluorescence probes usually remain in theexcited state from 1 to 100 nanoseconds (ns), a duration called thefluorescence lifetime. Because rotational diffusion of polypeptides alsooccurs in 1-100 ns, fluorescence lifetimes are a favorable time scalefor studies of the associative and/or rotational behavior ofmacromolecules. Other probes such as probes with lifetimes of several100 microseconds include, but are not limited to: [Ru(bpy)₂dcbpy)]²⁺R,where bpy is bispyridine, dcbpy is 4.4′ dicarboxyl bpy;[Os(bpy)₂(dcbpy)]²⁺; and (L)Re(CO)₃C=NR, where L is 1,10 phenanthrolineor bpy, R is n-Bu or t-Bu, and Bu is Butyl. A review of these complexesand references to them can be found in Terpetsching, et al., (Meth.Enzymol. (1997) 278:295). Thus, when a sample of a lipid matrix-embeddedmembrane polypeptide comprising an appropriate donor-acceptorchromophore pair is illuminated with vertically polarized light, theemission can be polarized. When energy transfer occurs between the samemolecules in identical environments, fluorescence intensity or lifetimedoes not change. The anisotropy on the other hand may change due tolikely differences in spatial orientation between themembrane-polypeptide and ligand attached chromophores.

Of particular interest for anisotropic assays is the ability torotationally restrict chromophores along their linker arms by providingtwo attachment sites such as two residues spaced by three interveningresidues along an α-helix. Such a design prevents attached chromophoresfrom freely rotating around the linker arm and thus prevents a loss ofspatial information from anisotropy assays. If a polypeptide binds aligand, the membrane polypeptide-bound and the ligand-chromophore (andtheir respective dipoles) have a defined spatial orientation withrespect to one another. More importantly, their relative orientationundergoes little fluctuation if the chromophore is rigidly attached.Since the signal-to-noise ratio of an anisotropy-detected experiment isa direct function of the extent of orientational fluctuation, rigidattachment of the chromophore allows one to detect FRET via anisotropywith a higher sensitivity.

In another embodiment of the invention, methods and compositions areprovided for labeling polypeptides with chelator-sensitizedtime-resolved metal ion probes, such as coumarin-sensitizedtime-resolved lanthanide probes. This aspect of the invention providesmethod and compositions for site-specific labeling of any peptide orpolypeptide amenable to solid-phase chemical synthesis or a combinationof solid-phase chemical synthesis and chemical ligation techniques,including soluble and membrane peptides and polypeptides, with asensitized zwitterionic metal ion chelator complex (MCC) for detectionof time-resolved luminescence in FRET and high throughput screeningassays. The method involves labeling one or more amino acids of apeptide or polypeptide chain that is attached to a chemical synthesisresin with a zwitterionic chelator moiety capable of chelating metalions. Thus, a chelator moiety is attached to the peptide or polypeptidechain concurrent with on-resin chemical synthesis. The metal ions maythen be complexed with the chelator moiety on-resin, or after thepeptide or polypeptide chain is cleaved from the resin, depending on itsintended end use. Chelator moieties suitable for this method include,but are not limited to, zwitterionic chelating agents from the group ofTTHA (triethylenetetraminehexaacetic acid), DTPA(diethylenetriaminepentaacetic acid), DTTA (diethylenetriamine N,N,N,Ntetraacetic acid), DOTA (1,4,7,10 tetraazacyclododecanceN,N′,N″,N′″tetraacetic acid), TETA (1,4,8,11 tetraazacyclotetradecaneN,N′,N″,N′″ tetraacetic acid) and any other zwitterionic chelatorligands for binding metals. Metal ions of particular interest arelanthanide ions, and more particularly Terbium (Tb³⁺), Samarium (Sm³⁺),and Europium (Eu³⁺).

Also provided is a method to increase the solubility of zwitterionicchelating agents including, zwitterionic chelating agents from the groupof TTHA, DTPA, DTTA, DOTA, TETA and any other zwitterionic chelatorligands for binding metals. This aspect of the invention involvescombining an insoluble zwitterionic chelating agent with a solubilizingagent that produces a soluble salt form of the zwitterionic chelatingagent. This method is applicable for any zwitterioni chelating agenthaving insufficient solubility in commonly used solvents such asdimethylformamide (DMF), dimethylsulfoxide (DMSO), methanol and thelike. In a preferred embodiment, the zwitterionic chelating agent to besolubilized comprises a carboxyl group that is to be activated for agiven subsequent coupling reaction, and the solubilizing agent is inertto activation. The preferred solubilizing agent is trifluoroacetic acid(TFA). For example, combination of the zwitterionic chelator TTHA withTFA yields the TFA salt of TTHA that can be prepared and stored as alyophilized powder that very readily dissolves in DMF, which is commonlyemployed in on-resin chemical synthesis. Another example is of asolubilizing agent is para-toluene sulfonic acid (TSA). Compared to TFA,the TSA has the advantageous properties of not only being a strong acid,but it also is non-volatile and non-nucleophilic. It will be appreciatedthat combinations of solubilizing agents also may be employed dependingon the intended end use. By employing the soluble salt of a zwitterionicchelator in on-resin labeling of peptides or polypeptides, the reactionyields are significantly improved by increasing the concentration of thesoluble chelator available for reaction. Also provided is a compositioncomprising a soluble salt form of a zwitterionic chelator. The preferredsoluble salt form of a zwitterionic chelator is TFA salt of azwitterionic chelator. Another soluble salt form of a zwitterionicchelator is TSA salt of a zwitterionic chelator. This includes, but isnot limited to, TFA and TSA salts of zwitterionic chelating agents fromthe group of TTHA, DTPA, DTTA, DOTA, TETA and any other zwitterionicchelator ligands for binding metals.

On-resin labeling of peptides and polypeptides with chelator-sensitizedtime-resolved metal ion probes permits site-specific labeling, highpurity by removal of residual reaction components and dye, andunprecedented yield. This avoids previous problems in which a peptide orpolypeptide is labeled after recombinant production or labeling afterassembly of the chain by chemical synthesis. For instance, labeling offolded peptide or polypeptide with dye or other label often causesnon-specific, non-covalent binding of the label. In contrast, with theon-resin chelator labeling method of the invention it is much easier tocontrol and characterize where the label is positioned using solid-phaseattachment. This is because all sites of potential side-reactions areprotected on the resin as opposed to unprotected in traditional labelingof folded peptides or polypeptides; the lipid matrix-assisted chemicalligation approach of the invention also can provide this advantage, suchas when chelator complex preparation is utilized in conjunction withchemoselective hydrazone or similar attachment. Solid phase synthesisand on resin labeling of the method also is followed by multiple washeswith organic solvent, minimizing (essentially eliminating) any residualdye or other label that could non-covalently bind. With regard to yield,prior approaches of post-resin labeling with lanthanide ion chelatorcomplexes typically provide a reaction yield of about 10-50% forcomplexation involving coupling of sensitizor to dye (e.g., coupling ofAMCA-X or carbostyril cs124 (7-amino,4-methyl-2(l H)-quinoline) to TTHAor DTPA (diethylenetriaminepentaacetic acid) sensitized complex (TTHAyield reported to be about 10-25%, and DTPA yield reported to be about40-50%); see, e.g., Heyduk, et al., Anal. Biochem. (1997) 248:214; Li,et al., Am. Chem. Soc. (1995) 116:8132; and Mathis, et al., Clin. Chem.(1995) 41:1391). By way of contrast, the on-resin labeling method of thepresent invention is basically complete (>95%). This extraordinarydifference in yield is due in part to (1) the inherent advantage ofsolid-phase synthesis in a gel material that perfectly solvatesreagents, and (2) the ability to achieve high concentration of chelator(e.g., TTHA) in DMF by prior conversion of chelator to the TFA salt.Other advantages include its ease of preparation. For example,attachment of the label on-resin takes a few steps of solid-phasesynthesis. All following steps such as ligation, purification andfolding are identical as for native protein. In contrast, labeling offolded protein requires potentially extensive purification work toremove unreacted material.

The methods and compositions of the invention have tremendous potentialfor therapeutic applications, as they can be used in diagnostic assaysas well as in drug discovery, for example, in high throughput screeningof compound libraries that contain ligands for membrane polypeptides ofinterest. The invention is widely applicable for synthesis andstructure/function study of folded membrane polypeptides, includingsingle-pass and multi-pass membrane polypeptides. This includes thedetectable labeling and modular assembly of membrane polypeptides fromseparately synthesized and folded extra-membranous and transmembranedomains. Furthermore, recombinant polypeptides that include a native orengineered cleavage site adjacent to a targeted chemoselective ligationsite residue can be cleaved and ligated to almost any desired peptideusing a semi-synthetic approach. Thus, the present invention allows forthe incorporation of a vast variety of molecular tools such asspectroscopic probes, unnatural amino acids and molecular markers atyields that significantly exceed those achieved with currently availabletechniques.

Exploiting the physical and chemical properties of a lipid matrix incombination with the diversity, specificity and yield of chemoselectivechemical ligation techniques provides access to virtually any membranepolypeptide comprising one or more suitable chemoselective ligationsites, whether the sites are engineered and/or occur naturally. Thisincludes access to single or multiple transmembrane domains connected toextra-membranous domains, such as N-terminal and C-terminal sequences,and/or internal loops that extend out of the membrane bilayer. As anexample, extra-membrane loops can be shortened, cut, or elongated withsegments forming proteolytic cleavage sites, foreign epitopes,extra-transmembrane segments, or even whole proteins systems, forexample, to facilitate purification, biochemical/biophysical studies, orcrystallogenesis. Transmembrane segments, such as α-helices, also can bedeleted, duplicated, exchanged, transported into a foreign context orreplaced with synthetic peptides, in order to characterize theirintegration into, and assembly in, the membrane and their function.Insertion of residues that serve as a subsequent target of achemoselective ligation chemistry of interest also can serve as thebasis for a great diversity of experiments, ranging from the explorationof secondary, tertiary and quaternary structures of the transmembraneregion to the creation of anchoring points for reporter molecules thatare joined to a prefolded membrane polypeptide through a ligation labelcomprising a covalent bond at the ligation site. Since the methods andcompositions of the invention are amenable to chemical synthesis, andgiven the small size of many folding domains in membrane polypeptides,particularly α-helical domains, modular synthesis of multi-pass membranepolypeptides is possible.

Moreover, methods and compositions of the invention can be exploited forresonance energy transfer measurements by FRET analyses. This includesaccess to donor-acceptor chromophore systems that can be used as aqualitative or a quantitative tool to detect and characterizeinteractions between a folded lipid matrix-embedded membrane polypeptideand a ligand for the polypeptide, such as a membrane boundreceptor-ligand system. The principles and applications of employingresonance energy transfer systems are many and well known, and thus arereadily exploited using the methods and compositions of the invention.For instance, lipid matrix-assisted chemical ligation and synthesis canbe exploited to create a chromophore donor/acceptor system that enablesdetection through FRET. Since measurement of energy transfer is based onfluorescence detection, the assays are highly sensitive and can be usedto detect ligand binding when the labeled membrane polypeptide is areceptor for the ligand. Additionally, atomic-structural information formembrane polypeptides can be obtained regardless of the complexity orheterogeneity of the system. Since the time scale of resonance energytransfer can be on the order of nanoseconds to microseconds, manyprocesses including slow conversion of conformers that are time-averagedin other techniques can be resolved. This approach can be used to inferthe spatial relation between donor and acceptor chromophores to obtainstructural information, including ligand-induced conformational changes.In addition to data acquisition with a conventionalspectrophotofluorometer, the FRET methods can be adapted for multiple invitro and in vivo assays including liquid chromatography,electrophoresis, microscopy, and flow cytometry etc. Thus, the presentinvention can be used for both in vitro and in vivo assays. The methodsalso can be applied as a simple diagnostic tool, as well as used in thestudy of membrane structure and dynamics, or extend it to molecularinteractions on cell surfaces or in single cells.

Lipid matrix-assisted chemical ligation and membrane polypeptidesynthesis also can be exploited for “D-target” screening. Many drugs aretargeted at integral membrane polypeptides, such as receptors,particularly G-protein coupled receptors, and ion channels. Such drugsare typically either chiral small molecules often natural-productderived (usually receptor antagonists), or chiral {peptide, polypeptide,or carbohydrate} molecules. These latter biomolecular ligands can haveeither agonist or antagonist effects on the integral membranepolypeptide depending on the properties of a particular system. See, forexample, Kent, et al., WO 93/25667.

All naturally occurring polypeptides are made up exclusively of L-aminoacids plus the achiral amino acid glycine. (Very rarely an amino acidmay be chirally inverted to the opposite “D-” configuration bypost-translational enzymatic action.) Thus, natural polypeptides aremade up of polypeptide chains exclusively of the L-configuration. Thechiral polypeptide chain folds to form a defined three dimensionalstructure characteristic of a particular polypeptide molecule. It isthis folded three-dimensional form that gives a polypeptide its specificproperties such as binding, enzymatic activity, transporter function,etc.

The particular folded shape of a polypeptide is determined by thesequence and chirality of the amino acids in the polypeptide chain. Ithas been shown experimentally that a polypeptide chain of oppositechirality i.e. one made up of D-amino acids [inverted stereochemistry atall chiral centers including the side chain Cβ atoms of Thr and Ile],will fold to form a polypeptide molecule with a shape that is the mirrorimage of the polypeptide molecule formed by folding the correspondingL-polypeptide chain. Furthermore, such a “D-polypeptide” will havereciprocal chiral ligand-binding properties compared with thecorresponding L-polypeptide, i.e. from a mixture of enantiomers, theL-polypeptide will bind one enantiomer of a compound, while theD-polypeptide will bind the other enantiomer of the same compound.

These reciprocal binding properties of mirror image properties can betaken advantage of in the discovery of new drug leads i.e. unique chiralsmall molecules that bind to a particular polypeptide and interfere withits biological function. Thus, a mixture of small molecules, ofarbitrary chirality and consisting of only a single enantiomer of eachmolecule, such as a mixture of natural product compounds isolated frommicrobial, plant, or other natural sources, can be used for screeningfor binding molecules using the mirror image, i.e. D-form of a membranepolypeptide produced using lipid matrix-assisted chemical ligation andsynthesis. Chiral molecules identified as binding molecules by thisprocess will bind only to the D-polypeptide and will not bind thecorresponding L-polypeptide. However, if the chiral molecule(s)identified as binding molecules for the D-polypeptide are reproduced(synthesized) as the identical molecule but of opposite chirality i.e.the other enantiomer that was not present in the original compoundmixture used for screening, then that newly synthesized enantiomer willnot bind the D-polypeptide used for screening, but will bind thecorresponding L-polypeptide.

In this way, unique chiral binding molecules for natural polypeptidedrug targets can be discovered in libraries of chiral (natural product)molecules. Such binding molecules could not be discovered using thecorresponding natural polypeptide of L-configuration.

Lipid matrix-assisted membrane polypeptide chemical ligation accordingto the method of the invention also can be utilized to gain facileaccess to integral membrane proteins that are anchored to the lipidbilayer by covalent attachment of hydrophobic moieties such as fattyacids, prenyl groups or glyosylphosphatidylinositol anchors. Althoughmany such polypeptides themselves are soluble, attachment of thesehydrophobic moieties may reduce the solubility of these proteins andrender them difficult to work with in a cell free environment.Accordingly, performing chemical ligation in the presence of a lipidicphase facilitate synthetic access to the acylated forms of theseproteins that are inaccessible by other approaches.

Fatty acid chains, such as myristyl acid, can be site-specificallyattached to the N-terminus of a peptide on resin through an amide bond.Prenyl groups and palmitic acid groups can be coupled to specificcysteine residues on resin through thioether linkages. The peptide canthen be cleaved off the resin in HF, yielding an unprotected peptidewith a covalently attached fatty acid or lipid chain.

The following examples illustrate various aspects of this invention.These examples do not limit the scope of this invention.

Abbreviations AGRP Agouti-Related Protein AMCA-X6-((7-amino-4-methylcoumrin-3-acetyl)amino)hexanoic acid Boctert-butoxycarbonyl CLP Cubic Lipidic Phase matrix DIEAdiisopropylethyleamine DMF N,N-dimethylformamide DMSO dimethylsulfoxideDNP dinitrophenyl DOPC Dioleoyl phosphatidyl choline DPCdodecylphosphocholine EDTA Ethylene diamine tetraacetic acid,tetrasodium salt ES/MS electrospray mass spectrometry Fmoc9-fluorenylmethoxycarbonyl FRET Fluorescence Resonance Energy TransferHBTU O-(1H-benzotriazol-1-yl)-1,1,3,3 -tetramethyl-uroniumhexafluorophosphate HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid HF hydrogen fluoride hIL-8 Human Interleukin 8 HPLC HighPerformance Liquid Chromatography IRK1 Inward-Rectifying Potassiumchannel pore KCSA potassium ion channel pore of Streptomyces lividansLUV Large Unilamellar Vesicles MALDI matrix assisted laser desorption(mass spectrometry) MBHA methylbenzhydrylamine MC4 melanocortin MO1-monooleoyl-rac-glycerol (C18:1, {cis}-9) OG β-octyl glucopyranosidePAM phenyl(acetamido)methyl, i.e. - OCH₂C₆H₄CH₂CONHCH₂C₆H₄-(polystyrene)SDS-PAGE Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis TAMRAcarboxytetramethylrhodamine TCEP triscarboxy ethyl phosphine TFAtrifluoroacetic acid TFE trifluoroethanol TTHAtriethylenetetraminehexaacetic acid

EXAMPLES Example 1 Design and Synthesis of Bacteriorhodopsin MembranePolypeptide

Bacteriorhodopsin functions as a light-driven proton pump in the purplemembrane of halobacteria and has a characteristic seven helicalstructural motif. It is part of a family of retinal-binding proteinsthat includes the mammalian vision receptor, rhodopsin.Bacteriorhodopsin can be refolded from a fully denatured state into afunctional, native protein, and its atomic structure is known. Segmentsof bacteriorhodopsin can also be designed which independently form amembrane bilayer spanning α-helix when incorporated into a lipidmembrane (Hunt, et al., Biophysical Journal (1991) 59:400a).

A membrane polypeptide derived from the first transmembrane α-helix ofbacteriorhodopsin and its first extracellular loop (corresponding tobacteriorhodopsin residues 5-42) is selected for native chemicalligation. The membrane polypeptide is modified so that the N-terminalThr residue (corresponding to Thr5 of bacteriorhodopsin) is replacedwith a Cys residue to serve as ligation site for native chemicalligation (Dawson, et al., Science (1994) 266:776-779).

This Cys residue is capped with an N-terminal Factor Xa proteasecleavage site and a few random residues to test for the ability toobtain a free N-terminal cysteine residue by protease cleavage. To easepeptide purification and detection for MALDI-analysis, an imino-biotinlabel is attached to the C-terminal Lys residue (corresponding to Lys41of bacteriorhodopsin). Unless otherwise stated, all amino acid sequencesare provided in the N-terminal to C-terminal direction.

Bacteriorhodopsin membrane polypeptide 1: Native bacteriorhodopsinα-helix 1 and first extracellular loop (SEQ ID NO:1): TGRPEWIWLALGTALMGLGT LYFLVKGMGV SDPDAKK. Bacteriorhodopsin membrane polypeptide 2:Cys-modified bacteriorhodopsin α-helix 1 and first extracellular loop(SEQ ID NO:2): CGRPEWIWLA LGTALMGLGT LYFLVKGMGV SDPDAKK.Bacteriorhodopsin membrane polypeptide 3: Factor Xa protease cleavagesite, Cys-modified bacteriorhodopsin α-helix 1 and first extracellularloop (SEQ ID NO:3): GKGYIEGRCG RPEWIWLALG TALMGLGTLY FLVKGMGVSD PDAKK

Bacteriorhodopsin membrane polypeptide 3 (SEQ ID NO:3) is synthesized onan MBHA resin using a in situ neutralization protocol for Boc chemistryusing established side-chain protection strategies except that theγ-amino group of the C-terminal Lys is protected with an Fmoc group(Schnolzer, et al., Int. J. Pept. Protein Res. (1992) 40:180-193).Machine-assisted peptide synthesis is performed on a custom-modifiedApplied Biosystems 430A peptide synthesizer following establishedprotocols (Schnolzer, et al., supra). The Fmoc protecting group of theC-terminal Lys is removed after peptide synthesis by incubation with 20%piperidine in DMF and an N-imino biotin group is attached to the freeamino group by addition of 2-imino N-hydroxysuccinimide (Sigma, St.Louis). The peptide is deprotected and simultaneously cleaved from theresin by treatment with HF in the presence of 10% v/v p-cresol for 1hour at 0° C. The crude peptide is precipitated in diethyl ether, takenup in 75% B (acetonitrile+0.1% TFA) and lyophilized. Polypeptide 3 (SEQID NO:3) is purified by preparative reversed-phase HPLC (45-75% B over60 minutes at 10 ml/min) and characterized by electrospray MS (obs. MW5080±1 kD, calc. 5080 kD (average isotope composition)). Gradient HPLCis performed on a Rainin dual-pump high-pressure mixing system with 214nm UV detection using a Vydac C-4 semipreparative column (10 μm particlesize, 1 cm×25 cm) or a Vydac C-4 preparative column (10 μm particlesize, 2.2 cm×25 cm). Electrospray mass spectra is obtained using a SciexAPI-1 quadrupole ion-spray mass spectrometer. MALDI mass spectra isobtained using a ciphergen xyz TOF-MALDI instrument.

Example 2 Design and Synthesis of Ligation Label

A C-terminal thioester-modified ligation label peptide EAQL (SEQ IDNO:4) designed for native chemical ligation to the lipidmatrix-incorporated membrane polypeptide (SEQ ID NO:2) is synthesizedmanually on a PAM-thioester generating resin, cleaved and deprotected asdescribed above. The crude peptide is precipitated in diethyl ether,taken up in 50% B and lyophilized. The peptide is purified by semipreparative reversed-phase HPLC (25-45% B over 45 minutes at 10 ml/min)and characterized by electrospray MS (obs. MW 647±1 kD, calc. 647(average isotope composition)).

Example 3 Preparation of CLP Matrix and Incorporation ofBacteriorhodopsin Membrane Polypeptide

Cubic lipidic phase matrices admixed with membrane polypeptide 3 (SEQ IDNO:3) are prepared following standard protocols by centrifugation of thelipid matrix constituents in a Haraeus Biofuge 13 table-top centrifuge.In particular, 75 μg of membrane polypeptide 3 (SEQ ID NO:3) isdissolved in 3 μl 200 mM Tris buffer containing 2% OG at pH 8. Thesepolypeptide solutions are added to a 1.7 ml eppendorf tube containing14.2 mg MO. Subsequently, 1.5 μl 200 mM Tris buffer (pH 8) is added todecrease the lipid:water ratio. The mixture is spun for 3 hours at12,000 rpm at approximately 25° C. Formation of CLP's is indicated bythe formation of optically clear, gel-like phases. These phases remainoptically clear at room temperature for at least two months.Off-resonance Raman spectra excited at 514.5 nm is used to demonstratethat the addition of the membrane polypeptide does not perturb themembrane bilayer structure of the CLP, as indicated by an unalteredintensity ratio of the C-H stretching modes at 2885 and 2845 cm⁻¹ in theabsence and presence of membrane polypeptide in the CLP (Razumas, etal., Chemistry and Physics of Lipids (1996) 84:123-138).

Example 4 Affinity Purification of CLP Matrix-IncorporatedBacteriorhodopsin Membrane Polypeptide and MALDI-TOF Analysis

CLPs (MO) containing bacteriorhodopsin membrane polypeptide 3 (SEQ IDNO:3) are solubilized by adding 33 mg of OG in 100 μl of water. Toprevent unwanted ligation during analysis, 20% β-mercaptoethanol isadded during solubilization. The pH of the solubilized phase is adjustedto 9.5 using 0.2 M NaOH. Streptavidin-coated magnetic beads (Sigma, S2415) are added to the solubilized phases and incubated at roomtemperature for 1 hour. The magnetic beads are washed 5 times with 20 mMBis-Tris Propane buffer, pH 9.5, isolated by magnetic separation aftereach wash. The magnetic beads are resuspended in a saturated solution ofα-cyano-4-hydroxy-cinnamic acid in 50% acetonitrile/0. 1% TFA. A 1 mLaliquot of the magnetic beads/matrix suspension is deposited directly onthe sample slide and allowed to air dry. The sample preparations areanalyzed using a Ciphergen Biosystems Massphoresis System time-of-flightmass spectrometer. All spectra are internally calibrated using peptidestandards and represent an average of 25 shots.

Example 5 Protease Cleavage of CLP Matrix-Incorporated BacteriorhodopsinMembrane Polypeptide

Protease cleavage of the CLP matrix-incorporated bacteriorhodopsinmembrane polypeptide 3 (SEQ ID NO:3) is preformed by adding 1.5μl of a 1mg/ml solution of Factor Xa (New-England Biolabs) in 50% glycerol and 20mM HEPES, 50 mM NaCl, 2 mM CaCl₂ (pH 8.0) to the CLP. The mixture isthoroughly mixed using a plastic pipette tip and the CLP spun for 15minutes at 12,000 rpm to obtain formation of an optically clear phase.The extent of cleavage after incubation overnight and generation of CLPincorporated cleavage product is determined by MALDI mass spectrometryas described above.

Example 6 Native Chemical Ligation of CLP Matrix-IncorporatedBacteriorhodopsin Membrane Polypeptide

Native chemical ligation of CLP-incorporated polypeptide is performed asfollows. Approximately 10 μg of the ligation label peptide EAQL (SEQ IDNO:4) is dissolved in 1 μl of 200 mM Tris buffer (pH 8) and added to theCLP containing the Cys-terminal transmembrane polypeptide (SEQ ID NO:2),followed by addition of 0.5 μl thiophenol and 3 mg MO to maintain anappropriate lipid:water ratio compatible with CLP formation. The mixtureis mixed thoroughly using a small pipette tip. The CLP is then spun for15 minutes at 12,000 rpm to form an optically clear phase. Aliquots ofthe CLP are removed at specific time intervals to monitor the progressof ligation by MALDI mass spectrometry as described above. The primarychemical ligation reaction product is as follows.

Bacteriorhodopsin membrane polypeptide 5: Chemical ligation labeled,Cys-modified bacteriorhodopsin α-helix 1 and first extracellular loop(SEQ ID NO:5): EAQLCGRPEW IWLALGTALM GLGTLYFLVK GMGVSDPDAK K.

FIG. 3 provides a schematic illustrating protease cleavage and use ofnative chemical ligation to generate a labeled CLP matrix-embeddedmembrane polypeptide. After incorporation into the CLP lipid bilayer,the N-terminal polypeptide containing the protease recognition site isremoved by Factor Xa cleavage (A). A ligation peptide corresponding tothe N-terminus of bacteriorhodopsin (Ile4 is replaced with Leu4 tooptimize ligation) is then ligated to the transmembrane polypeptide toobtain the full bacteriorhodopsin N-terminus (B). The extent of cleavageand the progress of the ligation inside the CLP is measured by obtainingMALDI mass spectra of the transmembrane polypeptide adsorbed to magneticbeads through a N-iminobiotin label (C).

FIG. 4 shows a typical MALDI mass spectra output that monitors creationof a free unprotected N-terminal cysteine by protease cleavage, followedby the chemical ligation of a ligation peptide of choice to thisresidue. Spectrum A presents a MALDI spectrum of polypeptide 3 (SEQ IDNO:3) after incorporation into a CLP, followed by dissolution of the CLPin OG/20% β-mercaptoethanol. The spectrum displays one major peak atm/z=5080, corresponding to the mass of the uncleaved polypeptide 3 (SEQID NO:3). Smaller peaks represent impurities from the affinity beads,and also are present in spectra in the absence of protein (data notshown). Spectrum B presents a mass spectrum of the same polypeptideafter cleavage with Factor Xa overnight. The disappearance of the majorpeak at m/z=5080 concomitant with the appearance of a major peak atm/z=4220 is consistent with almost complete removal of the Cys-cappingsequence GKGYIEGR by the Factor Xa protease and generation ofpolypeptide 2 (SEQ ID NO:2).

Spectra C, D and E monitor the appearance of ligation product afteraddition of a 2-fold excess of ligation peptide 4 (SEQ ID NO:4) with 5%thiophenol to the Cys-terminal membrane polypeptide 2 (SEQ ID NO:2)after 25 and 75 minutes and overnight incubation, respectively. Thegradual disappearance of the peak at m/z=4220 with a concomitantincrease of a peak at m/z=4661 demonstrates the progress of theformation of a native amide bond between the α-helical transmembranepolypeptide 2 (SEQ ID NO:2) and ligation peptide 4 (SEQ ID NO:4). Asjudged from the MALDI mass spectrum, the conversion from the Cys-cappedtransmembrane polypeptide to the fully ligated transmembrane polypeptide(SEQ ID NO:5) is completed to greater than 90%.

Example 6 Reverse Phase HPLC Purification of Membrane PolypeptideIncorporated in Cubic Lipidic Phase

Membrane polypeptide chemical ligation is performed with membranepolypeptide 1 (SEQ ID NO: 1) and membrane polypeptide 4 (SEQ ID NO:4) asdescribed above to yield CLP-incorporated membrane polypeptide 5 (SEQ IDNO:5). After ligation is complete, the entire CLP is dissolved in 50 μl85% aqueous isopropanol containing 50 mM formic acid. The solution isthen diluted with an equal volume of 50 mM aqueous formic acid.

A C18 reverse-phase HPLC column is equilibrated at 45%(6:3:1/isopropanol/acetonitrile/water including 0.1% TFA). 20 μl ofdissolved CLP are injected into the column and eluted isocratically at45% isopropanol/acetonitrile/water including 0.1 TFA. The ligatedmembrane polypeptide 5 (SEQ ID NO:5) is eluted after 10 minutesisocratic elution and is positively identified by ES/MS. MO is elutedisocratically between 16 and 20 minutes. Membrane polypeptide 5 (SEQ IDNO:5) is isolated by lyophilization.

Example 7 Location of Bacteriorhodopsin Membrane Polypeptide in CLPMatrix

The channels inside a CLP are filled with aqueous buffer, whereas at alipid:detergent ratio of 300: 1, excess detergent is absorbed into thelipid bilayer phase. Additionally, complete ligation also is observed inthe absence of detergent (data not shown). Peptide 1 is extremelyhydrophobic peptide and does not dissolve in 6M guanidinium HCl/200 mMphosphate (pH 7.5). It is therefore unlikely for significant amounts ofthis peptide to be dissolved in the aqueous channel. Furthermore, novisible aggregation of hydrophobic peptide is observed after prolongedspinning of the CLP's at 12,000 rpm after incorporation of 1 mg ofpeptide into a CLP of the same size. Taken together with the previousobservation by CD FTIR spectroscopies and proteolysis protectionexperiments that peptide 1 folds independently to an cc-helicaltransmembrane peptide inside liposomes, (Hunt, et al., BiophysicalJournal (1991) 59:400a), it is reasonable to interpret that the cleavageand ligation of peptide 1 is performed on a peptide incorporated intothe membrane bilayer.

The above Examples collectively indicate that cleavage of transmembranepeptide 3 (SEQ ID NO:3) and ligation of transmembrane peptide 2 (SEQ IDNO:2) and peptide 4 (SEQ ID NO:4) is indeed performed on a polypeptidethat is incorporated into the membrane bilayer to form a lipid-matrix inwhich transmembrane peptide 5 (SEQ ID NO:5) is properly and functionallyembedded.

Example 8 Chromophore Labeling and FRET Analyses of BacteriorhodopsinMembrane Polypeptide in CLP Matrix

A chromophore labeled CLP matrix-embedded bacteriorhodopsin membranepolypeptide is constructed and analyzed by FRET by first labelingbacteriorhodopsin membrane polypeptide 1 (SEQ ID NO:1) with TTHAcoumarin and labeling the thioester ligation label, peptide 4 (SEQ IDNO:4) with TEXAS RED.

Membrane polypeptide 1 (SEQ ID NO:1) and ligation label peptide 4 (SEQID NO:4) are synthesized on a PAM thioester generating resin andcarboxy-generating resin, respectively using an in situ neutralizationprotocol for Boc chemistry using established side-chain protectionstrategies. The transmembrane bacteriorhodopsin with acoumarin-lanthanide complex as follows.

Prior to the actual labeling step, the TTHA-TFA salt reagent isprepared; 100 mg TTHA is dissolved in 50 ml neat TFA for 4 hours at roomtemperature. The solution is dried on a Rotavap, taken up in 50% aqueousacetonitrile and lyophilized to yield a white powder that very readilydissolves in DMF.

Labeling of free amino groups on-resin: For coupling to the polypeptideN-terminus, N-terminally Boc protected peptide resin is equilibrated inDMF. The terminal Boc group is removed by treatment with TFA (2×1minute) and then washed with DMF, 10% DIEA in DMF and DMF. For couplingto a lysine residue, γ-Fmoc protected Boc-lysine is incorporated intothe target polypeptide chain during chain assembly. The Fmoc-group isremoved either by treatment with 20% piperdine in DMF (2×5 minutes) orby treatment with a two-fold excess of DBU(1,8-dioazabicyclo[5.4.0]undec-7-ene) for one minute for the targetpolypeptide on thioester-generating resin. The polypeptide is thenwashed with DMF.

ACMA-X-Chelating complex fluorescent probes: The N-terminally Bocprotected peptide resin is equilibrated in DMF. The Fmoc protectinggroup of Lysine 36 in membrane polypeptide 1 (SEQ ID NO:1) is removed bytreatment with 20% piperidine in DMF for 10 minutes. Then, 10 mg ofAMCA-X (˜2× excess) is dissolved in 150 μl DMSO and added to 0.01 mmoldrained peptide resin for 1 hours. Unreacted AMCA-X is washed away withDMSO and DMF. Ninhydrin tests before and after the coupling reactiondemonstrated >95% reaction efficiency. Approximately 0.03 mmol TTHA-TFAsalt was dissolved in an equimolar amount of 0.5 M HBTU solution in DMFcontaining ˜20% DIEA and coupled to the peptide resin overnight. Theresin is washed with DMF and dichloromethane and dried. HF-resincleavage and peptide purification are performed following standardprocedures (Schnoelzer, 1992, supra). For complexation with thelanthanide ions (Eu³⁺, Tb³⁺, or Sm³⁺) the peptide is dissolved in 50%acetonitrile containing a 3-fold excess aqueous lanthanide chloride.Metal incorporation is monitored by ES/MS analysis. Absorbance spectraof the complexed peptide showed an absorption maximum at 329 nm.

TEXAS RED fluorescent probe: The N-terminally Boc protected polypeptide(SEQ ID NO:4) resin is equilibrated in DMF and the Boc group removed bytreatment with TFA two times for 1 minute. The N-terminus is thenneutralized by treatment with 10% DIEA for 2×1 minute. 25 mg of TEXASRED (˜2× excess) is dissolved in 150 μl DMSO and added to 0.02 mmoldrained peptide resin for 1 hour. Unreacted dye is washed away with DMSOand DMF. Ninhydrin tests before and after the coupling reactiondemonstrated >95% reaction efficiency. HF-resin cleavage and peptidepurification are performed following standard procedures. (Schnoelzer,1992, supra).

Both polypeptides (SEQ ID NO: 1 and SEQ ID NO:4) are added to the CLPmatrix as described in Example 4 along with an aqueous buffer solutioncontaining three-fold excess of chelating metal (Eu³⁺, Tb³⁺, or Sm³⁺)and 1% thiophenol. After ligation, the lipid matrix containing theligated membrane polypeptide is transferred into a small cuvette andequilibrated to obtain an optically isotropic CLP matrix gel.Fluorescence energy transfer experiments are performed in a fluorolog 3spectrofluorometer exciting with a pulsed Xe flash lamp at 330 nm anddetecting after a 50 microsecond gate between 500 and 750 μm. FRETdistances are analyzed by comparison to the known distance between thelabeling sites in the bacteriorhodopsin crystal structure.

Example 9 Chemical Ligation of Liposome-Incorporated BacteriorhodopsinMembrane Polypeptide

For preparation of lipid vesicles, egg-lecithin is dissolved in 2:1chloroform/methanol, evaporated to form a dry film in a round-bottomflask and further dried under vacuum in a lyophilizer. The lipid issuspended in aqueous buffer (5 mM HEPES, 0.2 mM EDTA, pH 7.2) byvortexing for 30 s to form multilamellar liposome vesicles. LUV's areprepared from phospholipids after 10 freeze/thaw cycles by extrusionthrough polycarbonate membranes three times with 0.4 μm pore diameterand then ten times with 0.1 μm pore diameter. The solution is spun at500× g to pellet lipid aggregates and the liposomes are then pelleted at48,000× g.

Bacteriorhodopsin membrane polypeptide 3 (SEQ ID NO:3) is dissolved in aminimum amount of neat TFE and added to a suspension of egg-lecithinliposome vesicles in aqueous buffer (5 mM HEPES, 0.2 mM EDTA, pH 7.2) ata lipid:protein ratio of 1000:1. Incorporation of membrane polypeptideinto liposome lipid membrane is observed by monitoringfluorescence-emission of tryptophan above 300 nm. The proteoliposomesare pelleted at 48,000 g and frozen in aliquots at −80° C.

For chemical ligation of the lipid-embedded polypeptide, an aliquot ofproteoliposomes is suspended in 5 mM HEPES, 0.2 mM EDTA, pH 7.2 andtreated with 1/100^(th) molar equivalent of Factor Xa protease andligated as described in Examples 3 and 5-6 and ligation monitored as inExample 4.

Example 10 Chemical Ligation of Micelle-Incorporated BacteriorhodopsinMembrane Polypeptide

300 μg of membrane polypeptide 2 (SEQ ID NO:2) and 50 μg C-terminalα-thioester modified ligation polypeptide 4 (SEQ ID NO:4) are dissolvedin 100 μl 100 mM phosphate buffer (pH 7.5) containing 10% Ammonyx-LO(N,N-dimethyl amineoxide) and 1 μl thiophenol. The solution is stirredfor 24 hours at room temperature in an eppendorf tube. The progress ofthe ligation is monitored by analytical reverse-phase HPLC. Ligatedmembrane polypeptide is isolated using semi-preparative reverse-phaseHPLC and the identity of ligated product (membrane polypeptide 5 (SEQ IDNO:5)) is confirmed by mass analysis employing electrospray massspectrometry.

Example 11 Chemical Ligation of Cell Membrane-Incorporated MelanocortinReceptor MC4

Membrane polypeptide chemical ligation of recombinantly producedmelanocortin receptor MC4, embedded in native lipid membrane patchesisolated from cells, is performed as follows. HEK-293 cellsoverexpressing MC4 receptor containing an engineered Factor Xa cleavagesite (SEQ ID NO: 6) are constructed as follows, where the MC4 ReceptorMembrane Polypeptide w/Factor Xa Cleavage Site (SEQ ID NO: 6) has thefollowing amino acid sequence:

MVNSTHRGMH TSLHLWNRSS YRLHSNASES LGKGYIEGRC YEQLFVSPEV FVTLGVISLLENILVIVAIA KNKNLHSPMY FFICSLAVAD MLVSVSNGSE TIIITLLNST DTDAQSFTVNIDNVIDSVIC SSLLASICSL LSIAVDRYFT IFYALQYHNI MTVKRVGIII SCIWAACTVSGILFIIYSDS SAVIICLITM FFTMLALMAS LYVHMFLMAR LHIKRIAVLP GTGAIRQGANMKGAITLTIL IGVFVVCWAP FFLHLIFYIS CPQNPYCVCF MSHFNLYLIL IMCNSIIDPLIYALRSQELR KTFKEIICCY PLGGLCDLSS RY

The MC4 membrane polypeptide (SEQ ID NO:6) is designed for chemicalligation following Factor Xa cleavage to a C-terminal α-thioestermodified MC4 Receptor Ligation Label (SEQ ID NO:7) having the followingamino acid sequence:

MVNSTHRG MHTSLHLW NRSSYRLH SNASESLG KGYSDGG

The ligation label optionally is modified to contain othermodifications, such as the insertion of non-natural amino acids,isotopically labeled amino acids, or fluorescent probes attached atspecific sites. For example, N-terminus of the ligation label ismodified by coupling a TEXAS RED in DMSO as described in Example 8.

Eukaryotic HEK-293 cells overexpressing the MC4 receptor (SEQ ID NO:6)are constructed as follows. The coding region of the gene for the MC4receptor is subcloned into bacteriophage M13 for performing singlestranded site-directed mutagenesis (Sambrook, et al., supra).Oligonucleotide-directed mutagenesis (Kunkel, 1984; Zoller & Smith,1987), in vitro mutagenesis kit Muta-Gene T4® (BioRad; Hercules,Calif.), is utilized to generated the Factor Xa site (bacteriophage M 13vector containing the mutant receptor gene, pGRFN-MC4-8a). Presence ofthe Factor X cleavage site is confirmed by a single-stranded DNAsequencing (Sequenase Version 2.0 (Life Science; Cleveland, Ohio)). DNAencoding mutant MC4 receptors are subcloned into eukaryotic expressionvector pcDNA3.1 (Invitrogen, Carlsbad, Calif.) to create the MC4receptor expression construct pGRFN-MC4-8b. This construct istransfected into HEK-293 cells (ATCC, Manassas, Va.) following standardprotocols (LipfectAmine Reagent® (Life Technologies; Gaithersburg, Md.))to obtain cell line cGRFN-MC4-8a.

Membrane patches are isolated from cGRFN-MC4-8a cells expressing MC4receptor membrane polypeptide (SEQ ID NO:6). Cells containingapproximately 107 receptors per cell recovered from 10 cm culture plateare washed five times with PBS (PBS: 1.12 M NaCl, 2.6 mM KC1, 8.1 mMNa₂HPO₄, pH 7.4), and incubated in hypotonic buffer (1 mM Tris-HCl, pH6.8, 10 mM EDTA). Cells are disrupted by shearing them in a syringethree times through a 26-gauge needle. Disrupted cells are loaded onto astep-density gradient and spun at 20,000× g for 10 minutes. The gradientinterface containing the membrane patches is taken up with a Pasteurpipette. The membrane patches are sedimented at 48,000× g for 10 minutesand washed three times followed by sedimentation. All procedures areperformed at 4° C. unless stated otherwise.

For cleavage of MC4 membrane polypeptide (SEQ ID NO:6) with Factor Xaand native chemical ligation to the MC4 receptor ligation label (SEQ IDNO:7), membrane patches containing recombinant MC4 receptor (SEQ IDNO:6) are suspended in 50 μl of 100 mM Tris/pH 8 containing proteaseinhibitors that are not targeting Factor Xa protease such as aprotinin,2 mg/mL, N-[N-(L-3-Trans-carboxirane-2-carbonyl)-L-leucyl]-agmatine, 10μg/mL, and pepstatin A, 2 mg/mL. 2 μl Factor Xa solution (1.0 mg/ml) isadded and the suspension incubated overnight at room temperature (˜25°C.). The membrane patches are sedimented at 48,000× g and washed threetimes with 100 mM phosphate buffer (pH 7.5). Cleavage of MC4 (SEQ IDNO:6) with Factor Xa yields the MC4 Receptor Membrane Polypeptide FactorXa Cleavage Product (SEQ ID NO:8):

CYEQLFVSPE VFVTLGVISL LENILVIVAI AKNKNLHSPM YFFICSLAVA DMLVSVSNGSETIIITLLNS TDTDAQSFTV NIDNVIDSVI CSSLLASICS LLSIAVDRYF TIFYALQYHNIMTVKRVGII ISCIWAACTV SGILFIIYSD SSAVIICLIT MFFTMLALMA SLYVHMFLMARLHIKRIAVL PGTGAIRQGA NMKGAITLTI LIGVFVVCWA PFFLHLIFYI SCPQNPYCVCFMSHFNLYLI LIMCNSIIDP LIYALRSQEL RKTFKEIICC YPLGGLCDLS SRY

Chemical ligation of cleaved MC4 (SEQ ID NO:8) in membrane patches to aMC4 ligation label (SEQ ID NO:7) with or without AMCA-X/TTHA/Eu³⁺isperformed as follows. A 2 mM solution of MC4 ligation label (SEQ IDNO:7) containing a C-terminal thioester is added in 1.5 M guanidiniumchloride/100 mM sodium phosphate (pH 7.5) containing 1% thiophenol andincubated overnight. Fluorescence labeled or native MC4 ligation label(SEQ ID NO:7) containing a C-terminal thioester is then added in 1.5 Mguanidinium chloride/100 mM phosphate (pH 7.5) containing 1% thiophenol.Labeling with AMCA-X/TTHA/Eu³⁺chelate is performed as described inExample 8, with the following modifications. After overnight incubation,the modified MC4 receptor-containing membranes are washed with PBS andaliquots are frozen at −80° C. The ligation product has the followingamino acid sequence (SEQ ID NO:9) corresponding to the semi-syntheticlipid matrix-embedded MC4 receptor, with or without the site specificfluorescence label:

MC4 Receptor membrane polypeptide-ligation label ligate (SEQ ID NO:9):

MVNSTHRGMH TSLHLWNRSS YRLHSNASES LGKGYSDGGC YEQLFVSPEV FVTLGVISLLENILVIVAIA KNKNLHSPMY FFICSLAVAD MLVSVSNGSE TIIITLLNST DTDAQSFTVNIDNVIDSVIC SSLLASICSL LSIAVDRYFT IFYALQYHNI MTVKRVGIII SCIWAACTVSGILFIIYSDS SAVIICLITM FFTMLALMAS LYVHMFLMAR LHIKRIAVLP GTGAIRQGANMKGAITLTIL IGVFVVCWAP FFLHLIFYIS CPQNPYCVCF MSHFNLYLIL IMCNSIIDPLIYALRSQELR KTFKEIICCY PLGGLCDLSS RY

Membrane patches containing chemically ligated MC4 receptor (SEQ IDNO:9) are dissolved in 10% SDS-PAGE loading buffer and loaded onto a 10%SDS-PAGE gel are identified by Western blotting. Alternatively, thefluorescent probe-labeled MC4 receptor is visualized directly on thegel.

Example 12 FRET Analysis of Chromophore-Labeled Melanocortin ReceptorMC4

FRET analysis of cs124/TTHA/Tb⁺³ or AMCA-X/TTHA/Eu³⁺chelate-labeled MC4receptor is performed as follows. Membrane patches containing receptorscarrying cs124/TTHA/Tb⁺³ or AMCA-X/TTHA/Eu³⁺chelate are constructed asin Example 11 and are suspended in PBS in a 200 μl cuvette. MC4 receptorligand AGRP is labeled with TAMRA (paired with Tb⁺³ chelate) or TEXASRED (paired with Eu⁺³ chelate) following the procedure described inExample 11. The human AGRP sequence (SEQ ID NO: 10) is:

MLTAAVLSCAL LLALPATRGAQ MGLAPMEGIRR PDQALLPELPG LGLRAPLKKTT AEQAEEDLLQEAQALAEVLDLQ DREPRSSRRCV RLHESCLGQQV PCCDPCATCYC RFFNAFCYCRK LGTAMNPCSRT

TAMRA-labeled or TEXAS RED-labeled AGRP is titrated into the cuvettefrom a stock solution in PBS and fluorescence spectra are taken in aFluorolog 3 spectrofluorometer equipped with a Xe flash lamp. Emissionis detected after 50 us for 1 ms between 400 and 700 nm.

To screen for inhibitors of ligand binding, potential inhibitors areadded and the change in shape of the fluorescence spectrum is monitored.Alternatively, the decay of the fluorescence emission is monitored at543 nm for TAMRA or 614 nm for TEXAS RED. Replacement of the quenchingnatural ligand by the inhibitor increases the fluorescence lifetime atthis wavelength.

Example 13 Total Synthesis of the Inward-Rectifying Potassium ChannelPore IRK1 and FRET Analyses of Its Interaction with Toxin Ligand

Polypeptides for total chemical synthesis and chromophore labeling ofchannel pore IRK1 are designed based on the three-dimensional structureof the homologous potassium channel of Streptomyces lividans (Dole, etal., Science (1998) 280:69-77) and to accommodate toxin binding(MacKinnon, et al., Science (1998) 280:106). The polypeptides areconstructed so that the N-terminal domain entails the outer helix andthe C-terminal domain engenders the inner and pore helices. A glycineresidue is engineered into position 120 of IRK1 to ease chemicalligation. Polypeptides for stepwise synthesis and the ligation productof IRK1 are depicted in the following SEQ ID NOS:

IRK1 N-terminal domain polypeptide (SEQ ID NO:11): MTIFITAFLG SWFFFGLLWYAVAYIHKDLP EFHPSANHTG IRK1 C-terminal domain polypeptide (SEQ ID NO:12):CVENINGLTS AFLFSLETQV TIGYGFRCVT EQCATAIFLL IFQSILGVII NSFMCGAILA KISRPKTotal Synthetic/Ligated IRK1 polypeptide (SEQ ID NO:13): MTIFITAFLGSWFFFGLLWY AVAYIHKDLP EFHPSANHTG CVEAINSLTD AFLFSLETQV TIGYGFRCVTEQCATAIFLL IFQSILGVII NSFMCGAILA KISRPK

The N-terminus of IRK1 (76-120) (SEQ ID NO: 1) is synthesized on a PAMthioester generating resin using an in situ neutralization protocol forBoc chemistry using established side-chain protection strategies. ATTHA-coumarin label is attached to the N-terminus of the N-terminalpolypeptide following the procedures described above. The polypeptidesare deprotected and simultaneously cleaved from the resin by treatmentwith HF in the presence of 10% v/v p-cresol for 1 hour at 0° C. Thecrude polypeptide is precipitated and washed three times in cold ether,and dissolved in neat TFA. The TFA is removed on a Rotavap and theresidue taken up in neat TFE to form a clear solution. The TFE solutionis diluted 10× with 50% aqueous acetonitrile containing 0.1% TFA andlyophilized.

For purification, the crude polypeptide is taken up in neat TFE anddiluted with 2 equivalents of 6M guanidinium chloride containing 100 mMacetate (pH 4). The material is loaded onto an 1 inch ID C4 prep HPLCcolumn equilibrated at 45% buffer B (100% acetonitrile containing 0.1%TFA). After running the column isocratically at 45% C at 10 ml/min for15 minutes, the peptide is eluted in a gradient from 45% to 75% C in 60minutes at 10 ml/min. Fractions are collected every 5 ml and analyzedfor purity by ES/MS.

The C-terminus of IRK1 (120-186) (SEQ ID NO: 12) is synthesized on amidegenerating MBHA resin using an in situ neutralization protocol for Bocchemistry using established side-chain protection strategies. Thepeptides are deprotected and simultaneously cleaved from the resin bytreatment with HF in the presence of 10% v/v p-cresol for one hour at 0°C. The crude peptide is precipitated and washed three times in coldether, and dissolved in neat TFA. The TFA is removed on a Rotavap andthe residue taken up in neat TFE to form a clear solution. The TFEsolution is diluted 10× with 50% aqueous acetonitrile containing 0.1%TFA and lyophilized.

For purification, the crude polypeptide is taken up in neat TFE anddiluted with 2 equivalents of 6M guanidinium chloride containing 100 mMacetate (pH 4). The material is loaded onto a 1 inch ID C4 prep HPLCcolumn equilibrated at 65% buffer C (60% isopropanol, 30% acetonitrile,10% H₂O containing 0.1% TFA). After running the column isocratically at65% buffer C at 10 ml/min for 15 minutes, the peptide is eluted in agradient from 65% to 95% buffer C in 45 minutes at 10 ml/min. Fractionscollected every 5 ml are reconstituted with lanthanide ion chelate(e.g., terbium chelate Tb³⁺Cl₃) and analyzed for purity by ES/MS.

For lipid-matrix assisted membrane chemical ligation in CLP matrixforming lipid, 50 μg N-terminal polypeptide (SEQ ID NO: 11) and 60 μgC-terminal polypeptide (SEQ ID NO: 12) are dissolved in 3 μl DMSO andadded to 42.6 mg MO and 1 μl thiophenol in an Eppendorf centrifuge tube.The mixture is spun for 2.5 hours until formation of a clear phaseobserved. The reaction is left overnight. MALDI analysis shows thepresence of ligation product (SEQ ID NO:13) by mass analysis.

Agitoxin ligand (SEQ ID NO:14) designed to bind to the synthetic IRK1potassium channel is constructed by chemical synthesis taking intoconsideration the known channel-toxin interaction sites (Doyle, et al.,supra; Savarin, et al., Biochemistry (1998) 37:5407-5416). A TEXAS REDlabel is attached to the C-terminal lysine (residue 39) of agitoxin2(SEQ ID NO: 14) following the procedure described in Example 8. Thetoxin, IRK1 Agitoxin2 Ligand (SEQ ID NO: 14), has the following aminoacid sequence:

-   -   GVPINVSCTG SPQCIKPCKD AGMRFGKCMN RKCHCTPK

The IRK1 toxin peptide is purified by reverse phase HPLC and folded in2M guanidinium chloride/100 mM Tris, pH 8.6 containing 8 mM cystine/1 mMcysteine followed by preparative reversed-phase HPLC (25-45% B(iso-propanol/acetonitrile 2: 1) over 45 minutes at 10 ml/min.

Lyophilized IRK1 toxin is then mixed with the CLP matrix containing thelanthanide ion chelate labeled IRK1 membrane polypeptide (SEQ ID NO: 13)and spun for equilibration. The clear, red gel is then applied to asolid sample holder in a Spex-Fluorolog 3 Fluorimeter and the presenceof energy transfer is determined by obtaining the 330 nm excitedfluorescence spectrum of the gel between 450 and 600 nm. An aqueoussolution of a small-molecule antagonist is then mixed into the CLP anddisplacement of the toxin from the channel is observed by a decrease influorescence.

Example 14 Chemical Ligation of CLP-Incorporated Potassium Ion Channelof S. livdans.

Peptides utilized for the membrane spanning domains of the potassium ionchannel pore of Streptomyces lividans (KCSA) are designed based on theknown KCSA structure (Doyle, et al., Science (1998) 280:69-77). TheN-terminal transmembrane domain (SEQ ID NO:15) utilized for ligation isdesigned to entail the outer helix, whereas the C-terminal transmembranedomain (SEQ ID NO: 16) design engenders the inner and pore helices. Thisligation design yields a truncated version of the naturally occurringKCSA to isolate functionality of the membrane spanning domain formextracellular portions. Also, a cysteine is incorporated in place ofAla54 of the naturally occurring KCSA (160 residues) during peptidesynthesis to introduce a chemoselective group amenable to nativechemical ligation, for the purpose of generating a native peptidebackbone at the ligation site. Sequences of the N-terminal, C-terminaland final ligation product are depicted below.

KCSA Potassium Channel N-terminal Segment (SEQ ID NO:15): ALHWRAAGAATVLLVIVLLA GSYLAVLAER G KCSA Potassium Channel C-terminal Segment (SEQID NO:16): CPGAQLITYP RALWWSVETA TTVGYGDLYP VTLWGRLVAV VVMVAGITSFGLVTAALAT KCSA Potassium Channel Ligation Product (SEQ ID NO:17):ALHWRAAGAA TVLLVIVLLA GSYLAVLAER GCPGAQLITY PRALWWSVET ATTVGYGDLYPVTLWGRLVA VVVMVAGITS FGLVTAALAT

The N-terminus of KCSA (residues 23-53) (SEQ ID NO: 15) is synthesizedon a PAM thioester generating resin using an in situ neutralizationprotocol for Boc chemistry and established side-chain protectionstrategies. The peptides are deprotected and simultaneously cleaved fromthe resin by treatment with HF in the presence of 10% v/v p-cresol for 1hour at 0° C. The crude peptide is precipitated and washed 3× in coldether, and dissolved in neat TFA. The TFA is removed on a Rotavap andthe residue taken up in neat TFE to form a clear solution. The TFEsolution is diluted 10× with 50% aqueous acetonitrile containing 0.1%TFA and lyophilized. For purification, the crude peptide (SEQ ID NO: 15)is taken up in neat TFE and diluted with 2 equivalents of 6M guanidiniumchloride containing 100 mM acetate (pH 4). The material is loaded onto a1 in. ID C4 prep HPLC column equilibrated at 45% buffer C (60%isopropanol, 30% acetonitrile, 10% H₂O containing 0.1% TFA). Afterrunning the column isocratically at 45% buffer C at 10 ml/min for 15minutes, the peptide is eluted in a gradient from 45% to 75% buffer C in60 minutes at 10 ml/min. Fractions collected every 5 ml and analyzed forpurity by ES/MS.

The C-terminus of KCSA (residues 54-118) (SEQ ID NO:16) is synthesizedon amide generating MBHA resin using an in situ neutralization protocolfor Boc chemistry and established side-chain protection strategies. Thepeptide is deprotected and simultaneously cleaved from the resin bytreatment with HF in the presence of 10% v/v p-cresol for 1 hour at 0°C. The crude peptide is precipitated and washed 3× in cold ether, anddissolved in neat TFA. The TFA is removed on a Rotavap and the residuetaken up in neat TFE to form a clear solution. The TFE solution isdiluted 10× with 50% aqueous acetonitrile containing 0.1% TFA andlyophilized. For purification, the crude peptide (SEQ ID NO:16) is takenup in neat TFE and diluted with 2 equivalents of 6M guanidinium chloridecontaining 100 mM acetate (pH 4). The material is loaded onto a 1 inchID C4 prep HPLC column equilibrated at 65% buffer C. After running thecolumn isocratically at 65% buffer C at 10 ml/min for 15 minutes, thepeptide eluted in a gradient from 65% to 95% buffer C in 45 minutes at10 ml/min. Fractions collected every 5 ml and analyzed for purity byES/MS.

For incorporation into a CLP matrix, 50 mg of MO is dissolved in 2 ml(2:1 chloroform/methanol). 2 mg of DOPC is added to the solution. 70 μgN-terminal peptide (SEQ ID NO:15) and 90 μg C-terminal peptide (SEQ IDNO: 16) are dissolved in 150 μl TFE and heated to 60° C. for 30 minutesin a glass vial. Both solutions are then combined. The combined solutionis dried by blowing off solvent with argon gas and leaving under vacuumovernight to get a milky film. 35 μl Sodium phosphate (100 mM, pH 7.5)and 0.5 pl thiophenol are added to the dry film. The mixture is spun for2.5 hours until formation of a clear phase is observed. The reaction isleft overnight. The next day, 100 μl TFE containing 20%β-mercaptoethanol is added to the phase. The phase is briefly vortexedand spun in a tabletop centrifuge for 10 minutes. Then 150 μl buffer C(6:3:1 isopropanol/acetonitrile/H₂O containing 0.1% TFA) in 0.1% aqueousTFA is added. Residual precipitate is dissolved in neat TFA. Thesolution is filtered and a spatula tip of TCEP is added to reduce thesample. Finally, the dissolved reaction mixture is injected into a GPC(gel permeation chromatography) column filled with divinylbenzeneequilibrated in buffer C. The elutant peaks are collected and thefractions analyzed by MALDI mass spectrometry. (See FIG. 10).

Example 15 Chemical Ligation of Micelle-Incorporated HIV VPU Ion Channel

Total chemical synthesis of the HIV vpu ion channel utilizing nativechemical ligation is performed as follows. The N-terminal peptide (vpuresidues 1-39) (SEQ ID NO: 18): MEPIQLAIVA LVVAIIIAIV VWSIVIIYRKILRQRKID is synthesized on thioester-generating resin, and theC-terminal peptide (vpu residues 40-81) (SEQ ID NO: 19): CLIDRLIERAEDSGNESEGE ISALVEMGVE MGHHAPWDID DL, on standard resin-OCH₂ PAM. Thepeptides are combined by native chemical ligation at a non-nativecysteine residue suitably introduced during peptide synthesis andpositioned in the center of the protein. A 2 mM solution of theN-terminal peptide is prepared in a micelle-forming ligation buffercontaining 250 mM phosphate buffer (pH 7.5) containing 8 M urea, 250 mMDPC and 1 μl thiophenol. To this solution, a 1.2 fold molar excess ofC-terminal peptide is added. The solution is stirred for 3 hours at roomtemperature in an Eppendorf tube. The progress of the ligation ismonitored by analytical reversed-phase HPLC. The reaction is completedafter 3 hours, and represents greater than 95% yield of the initialreaction components. Ligated membrane polypeptide is isolated usingsemi-preparative reversed-phase HPLC and the identity of ligated productconfirmed by mass analysis employing electrospray mass spectrometry tohave a mass corresponding to expected sequence as follows (SEQ ID NO:20):

MEPIQLAIVA LVVAIIIAIV VWSIVIIYRK ILRQRKIDCL IDRLIERAED SGNESEGEISALVEMGVEMG HHAPWDIDDLSee FIG. 9.

Example 16 Chemical Ligation of Micelle-Incorporated Influenza M2 IonChannel

Total chemical synthesis of the influenza M2 ion channel is prepared asdescribed in Example 15 using the following M2 peptides formicelle-mediated ligation. The N-terminal peptide synthesized onthioester-generating resin has the following sequence(SEQ ID NO:21):

-   -   MSLLTEVETP IRNEWGSRCN DSSDPLVVAA SIIGILHLIL WILDRLFFK        which corresponds to M2 residues 1-49. The C-terminal peptide        synthesized on standard resin-OCH₂ PAM has the following        sequence (SEQ ID NO: 22): CIYRFFEHGL KRGPSTEGVP ESMREEYRKE        QQSAVDADDS HFVSIELE, which corresponds to M2 residues 50-97. As        with the HIV vpu ion channel, the reaction is completed after 3        hours with greater than 95% of the initial peptides being        ligated in the reaction to yield the ligated product (M2        residues 1-97). Semi-preparative reversed-phase HPLC and mass        analysis employing electrospray mass spectrometry confirmed a        ligation product having a mass corresponding to the expected        sequence as follows (SEQ ID NO:23):

MSLLTEVETP IRNEWGSRCN DSSDPLVVAA SIIGILHLIL WILDRLFFKC IYRFFEHGLKRGPSTEGVPE SMREEYRKEQ QSAVDADDSH FVSIELEAnalytical ultra centrifugation of the M2 ligation product incorporatedinto DPC micelles demonstrates that M2 oligomerizes into tetramers.

Example 17 Total Chemical Synthesis and On-Resin Labeling of Human IL-8Chemokines and FRET Analysis of Dimerization in Solution

Human IL-8 (hIL-8) is a member of the alpha (CXC) chemokine family andits dimerization properties have been well studied (Rajaratham, et al.(1997) Methods in Enzymology 287:89-105). The hIL-8 is synthesized andlabeled on-resin with a FRET pair to characterize dimerizationproperties in solution and for assays with its membrane polypeptidereceptors CXCR1 and CXCR2. A FRET pair TTHA-cs124-Tb³⁺label (donor) andTAMRA (acceptor) is an example of a labeling system chosen as suitablefor this purpose.

Peptides required for the total chemical synthesis and fluorescenton-resin labeling of human IL-8 are designed using the published aminoacid sequence of this protein. For synthesis and labeling design, anadditional lysine residue is added onto the C-terminus for on-resinattachment of the fluorescent probe, and a cysteine ligation site isselected in the middle of the sequence, resulting in two polypeptides of33 and 40 amino acids.

hIL-8 (1-33) N-terminal peptide (SEQ ID NO:24): SAKELRCQCI KTYSKPFHPKFIKELRVIES GPH hIL-8 (34-73) C-terminal peptide (SEQ ID NO:25):CANTEIIVKL SDGRELCLDP KENWVQRVVE KFLKRAENSK Synthetic full length hIL-8(1-73) (SEQ ID NO:26): SAKELRCQCI KTYSKPFHPK FIKELRVIES GPHCANTEIIVKLSDGRELC LDPKENWVQR VVEKFLKRAE NSK

The N-terminus thioester peptide of hIL-8 (1-33) is synthesized,deprotected and cleaved as described above, see e.g. Example 13. Topurify, the crude peptide is first dissolved in 6M guanidiniumchloride/0. 1 M sodium acetate, pH 4.0, then loaded onto a C4 reversephase preparative column equilibrated in 10% buffer B (100% acetonitrilecontaining 0. 1% TFA). The guanidinium was eluted by runningisocratically for 15 minutes at 10% buffer B at 13 mL/min. A gradient of30% to 50% in 60 minutes is used to elute off the peptide. Fractions arecollected every 6 mL, analyzed for purity by ES/MS, then lyophilized.

The acceptor-labeled C-terminal peptide hIL-8 (34-73-TAMRA) issynthesized on MBHA resin and labeled as described in Example 8 using5-(and-6)-carboxytetramethyl rhodarnine, succinimidyl ester. Thepolypeptide is then deprotected, cleaved and purified in a similarmanner to the N-terminal peptide. Donor labeled C-terminal peptidehIL-8-TTHA-cs124 is prepared as follows. The Fmoc side chain on lysine73 is removed by 20% piperidine (2×5 minutes) then washed well with DMF.10 Equivalents of the TFA salt of TTHA is dissolved in 0.5 M HBTU (1:1molar ratio of TTHA to HBTU), neutralized with 3.5 molar equivalents ofDIEA, then added to the resin. This is allowed to couple for 3 hours,until the ninhydrin looks clear. To couple the Cs 124 to the TTHAon-resin, one equivalent of HBTU per TTHA is added to the resin with 4equivalents of DIEA and allowed to activate for 1 minute. To theactivated resin, 10 molar equivalents of cs124 dissolved in DMF areadded and coupled for 3 hours. Excess cs124 is washed away with DMF andthe resin is cleaved in HF as described above. The labeled C-terminalpeptide hIL-8 (34-73-TTHA-cs124) is purified as described above.

Chemical ligation of the purified N-terminal peptide with each of thelabeled purified C-terminal peptides is performed in solution (6 Mguanidinium chloride/0. 1 M sodium phosphate, pH 7.0, 1 mM peptide) with0.1% thiophenol as a catalyst. Reaction is complete after 16 hours. Bothligation products are treated with β-mercaptoethanol (20%) at pH 8.6 for20 minutes to remove any remaining DNP protecting groups on thehistidine residues. The pH of the solution is dropped to 4.0, TCEP isadded to reduce any disulfides and the reaction mix is loaded on the C4reverse phase semi-prep column in 10% buffer B. Ligation product iseluted off using a gradient of 30 to 50% buffer B over 60 minutes.Fractions of 1.5 mL are collected, analyzed by ES-MS for purity, thenlyophilized.

The TAMRA-labeled hIL-8 polypeptide is folded by first dissolving thelyophilized ligation product in 6 M guanidinium chloride/0. 1 M Tris, pH8.0, then diluting down to 2 M with 0.1 M Tris, pH 8.0. Final peptideconcentration is 1 mg/mL. A redox buffer of 8 mM cysteine and 1 mmcystine is included to assist disulfide shuffling. After 16 hours, thefolding reaction is complete. This folded product is purified by RP-HPLCas described above. Fractions containing desired product are identifiedby ES-MS. A loss of 4 amu confirms the presence of two disulfide bridgesand the subsequent loss of 4 protons. The folded product is lyophilizedand stored at −20° C. until use. The TTHA-cs124 labeled hIL-8polypeptide is folded in a similar manner.

Folded hIL-8 (1-73-TTHA-cs124) is dissolved in 20 mM Bis Tris Propane,pH 6.5 and reconstituted with 3 equivalents of TbC13. The emissionspectra of the protein is collected using an excitation wavelength of342 nm and shows strong metal emission bands at 488, 543 and 583 nm,typical of Tb³⁺emission. Folded hIL-8 (1-73-TAMRA) is also dissolved in20 mM Bis Tris Propane, pH 6.5 and possesses a typical rhodamineemission centered at 577 nm when excited at 553 mm.

Distance between the donor and acceptor labels in a protein complex canbe determined from the measured fluorescent lifetimes according to thefollowing equations (Biological Spectroscopy by I.D. Campbell and R. A.Dwek (1984), Benjamin/Cummings Publishing Company, Inc. PP. 114-115.).The efficiency of energy transfer (E) between the two probes isdetermined by comparing the lifetime of the donor (TD) to the lifetimeof the donor in the presence of the acceptor (τ_(DA)) using thisequation, E=1−(τ_(DA)/τ_(D)). This efficiency is then related to theForster distance (R_(o)) to determine the average distance (R) betweenthe two labels using this equation, R=R_(o) {(1−E)/E} ⅙.

Equal concentrations (30 μM) of the donor, hIL-8 (1-73-TTHA-cs124-Tb³⁺),and the acceptor, hIL-8 (1-73-TAMRA) are mixed together and the lifetimeat 488 nm is measured. At this concentration, a large portion of theprotein will be dimerized in solution as the dimerization constant is˜10 μM. The decay curve exhibits bi-exponential decay. The majorcomponent of this decay possesses a shorter lifetime of 0.22 ms andcorresponds to the lifetime of the complex between donor and acceptor(τ_(DA)). This decrease in the fluorescent lifetime of the donoremission is due to fluorescence resonance energy transfer, FRET, fromthe donor to the acceptor. The minor component of the decay has alifetime of ˜1 ms and corresponds to the monomeric donor protein, or thedonor protein in complex with itself. The loss of emission over time at570 nm is also measured and shows a single phase decay with a lifetimeof 0.24 ms. At this wavelength, only the decay of the acceptor emissionresulting from FRET is observed. This lifetime is similar to thatdetermined at 488 nm. Finally, the decay of the fluorescence emission at488 nm of the donor alone (30 μM) is monitored over time afterexcitation at 342 nm. This decay curve exhibits mono-exponential decay,with a calculated lifetime of 0.94 ms (τ_(D)).

Using the measured lifetimes and the equations described above, anefficiency of energy transfer (E) of 75% and a distance (R) of ˜-50Å isobserved. This is a reasonable estimate of distance based on thepreviously determined NMR structure of the complex. The distance betweenthe two C-termini in this structure is 40Å. If the additional lysineside chains are considered, an estimate of 50Å seems reasonable. Theseresults demonstrate that the proteins are folded and forming dimers insolution. These results also further demonstrate the advantages ofemploying on-resin labeling for providing the specificity, purity andyield to conduct highly sensitive FRET analyses.

Example 18 Micelle-Mediated Chemical Ligation of emrE Transporter

Micelle-mediated chemical ligation is applied for the total chemicalsynthesis of the emre transport protein (SEQ ID NO: 27):

MNPYIYLGGA ILAEVIGTTL MKFSEGFTRL WPSVGTIICY CASFWLLAQT LAYIPTGIAYAIWSGVGIVL ISLLSWGFFG QRLDLPAIIG MMLICAGVLI INLLSRSTPH

The N-terminal emrE peptide segment 1 (SEQ ID NO: 28): MNPYIYLGGAILAEVIGTTL MKFSEGFTRL WPSVGTIICY, and middle emrE peptide segment 2 (SEQID NO: 29): C(Acm) ASFWLLAQT LAY AIWSGVGIVL ISLLSWGFFG QRLDLPAIIG MMLIare synthesized on thioester-generating resin and the C-terminal emrEpeptide segment 3 (SEQ ID NO: 30): CAGVLI INLLSRSTPH, on standard resinMBHA resin. 3.5 mg each of emrE peptide segments 2 thioester and emrEpeptide segment 3 are jointly dissolved in 50 μl TFE and 350 μl water.10 mg DPC powder are added. The clear solution is frozen in liquidnitrogen, and lyophilized for 5 hours, yielding a micelle matrix of DPCinterspersed with peptide. 400 μl 200 mM Phosphate buffer (pH 7.5)containing 8 M urea and 4 μl thiophenol are added to the lipid matrix,resulting in a clear solution of the peptide segments. After 2 days ofligation, the ligation mix is added to a solution of 2 ml TFE, 2 mlwater containing 20% β-mercaptoethanol and incubated for 20 minutes. Thesolution is acidified with a solution of 15 mg/ml TCEP in 20% aqueousacetic acid and loaded onto a semi-preparative reverse-phase HPLCcolumn. Fractions containing the desired first ligation product(ligation product of emrE segments 2+3) are identified by electrospraymass spectrometry and lyophilized overnight.

To remove the N-terminal “Acm” (acetamidomethyl) group, the resultinglyophilized/ligated emrE peptide segment (ligation product of emrEsegments 2+3, ˜2 mg) is dissolved in 50 μl TFE and 350 μl 0.5M aceticacid. The Acm group is then removed by treatment with a 1.5 molar excess(relative to the total cysteine concentration) of a Hg(acetate)₂solution for 1 hour. The solution is then made 20% in β-mercaptoethanol,loaded onto an analytical reverse-phase HPLC column and purified with astep gradient. Fractions containing the desired ligated product wereidentified by electrospray mass spectrometry and lyophilized overnight.

Equal amounts of the resulting deprotected ligated emrE peptide segment(ligation product of emrE segments 2+3) and the final emrE segment 1thioester are jointly dissolved in 50 μl TFE and 350 μl water. 5 mg DPCpowder is added. The clear solution is frozen in liquid nitrogen, andlyophilized for several hours, yielding a micelle matrix of DPCinterspersed with peptide. 200 μl 250 mM Phosphate buffer (pH 7.5)containing 8 M urea and 2 μl thiophenol are added to the lipid matrix,resulting in a clear solution of the peptide segments. After 1 day ofligation, the ligation mix is added to a solution of 1.4 ml TFE, 0.7 mlβ-mercaptoethanol, 0.7 ml piperidine and 1.4 ml water, and incubated for20 minutes to remove any remaining protecting groups. The solution isacidified with a solution of 15 mg/ml TCEP in 20% aqueous acetic acid,loaded onto a semi-preparative reverse-phase HPLC column and purifiedwith a linear gradient. Fractions containing the desired ligated product(SEQ ID NO: 27) are identified by electrospray mass spectrometry andlyophilized overnight.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference. The inventionnow being fully described, it will be apparent to one of ordinary skillin the art that many changes and modifications can be made theretowithout departing from the spirit or scope of the appended claims.

1. A composition for chemoselective ligation of a membrane polypeptide,comprising (i) a polypeptide comprising a first amino acid having anunprotected reactive group, wherein said polypeptide is incorporated ina lipid matrix; and (ii) a ligation label comprising a second amino acidhaving an unprotected reactive group, wherein said second amino acid iscapable of chemoselective chemical ligation with said first amino acidto form a covalent bond between said unprotected reactive groups of saidfirst and second amino acids under chemoselective chemical ligationconditions.
 2. The composition of claim 1 wherein said chemoselectivechemical ligation is selected from the group consisting of nativechemical ligation, oxime-forming ligation, thioester-forming ligation,thioether-forming ligation, hydrazone-forming ligation,thiazolidine-forming ligation, and oxazolidine-forming ligation.
 3. Thecomposition of claim 2 wherein the chemoselective chemical ligation isnative chemical ligation.
 4. The composition of claim 1 wherein saidlipid matrix comprises lipid molecules capable of forming a lyotropicphase.
 5. The composition of claim 1 wherein said lipid matrix isselected from the group consisting of a planner bilayer membrane, aliposome, a micelle, or a cubic lipidic phase matrix.
 6. The compositionof claim 1 wherein said membrane polypeptide is folded.
 7. Thecomposition of claim 1 wherein said membrane polypeptide is an integralmembrane polypeptide.
 8. The composition of claim 7 wherein saidintegral membrane polypeptide is a transmembrane polypeptide.
 9. Thecomposition of claim 8 wherein said transmembrane polypeptide is areceptor.
 10. The composition of claim 1 wherein said ligation labelcomprises one or more amino acids.
 11. The composition of claim 1wherein said ligation label comprises a peptide.
 12. The composition ofclaim 1 wherein the lipid matrix is an isolated lipid matrix.
 13. Thecomposition of claim 11 wherein said peptide is a membrane polypeptide.14. The composition of claim 11 wherein said peptide comprises anunnatural amino acid.
 15. The composition of claim 14 wherein saidunnatural amino acid comprises a chromophore.
 16. The composition ofclaim 15 wherein said chromophore is an acceptor moiety of anacceptor-donor resonance energy transfer pair.
 17. The composition ofclaim 15 wherein said chromophore is a donor moiety of an acceptor-donorresonance energy transfer pair.
 18. A composition comprising: i) aligation label comprising an amino acid having an unprotected reactivegroup; and ii) a folded polypeptide embedded in a lipid matrix, whereinsaid polypeptide comprises a cleavage site directly adjacent to an aminoacid capable of providing an unprotected reactive group upon cleavagewith a cleavage reagent and chemoselective chemical ligation with saidunprotected reactive group of said ligation label; wherein a covalentbond is capable of being formed between said unprotected reactivegroups.
 19. The composition of claim 18 wherein the lipid matrix is anisolated lipid matrix.